RESUMO
Over the past years, several studies have demonstrated that defects in DNA damage response and repair (DDR) genes are present in a significant proportion of patients with prostate cancer. These alterations, particularly mutations in BRCA2, are known to be associated with an increased risk of developing prostate cancer and more aggressive forms of the disease. There is growing evidence that certain DDR gene aberrations confer sensitivity to poly-(ADP ribose) polymerase inhibitors and/or platinum chemotherapy, while other defects might identify cases that are more likely to benefit from immune checkpoint inhibition. The potential prognostic impact and relevance for treatment selection together with the decreasing costs and broader accessibility to next-generation sequencing have already resulted in the increased frequency of genetic profiling of prostate tumours. Remarkably, almost half of all DDR genetic defects can occur in the germline, and prostate cancer patients identified as mutation carriers, as well as their families, will require appropriate genetic counselling. In this review, we summarise the current knowledge regarding the biology and clinical implications of DDR defects in prostate cancer, and outline how this evidence is prompting a change in the treatment landscape of the disease.
Assuntos
Dano ao DNA/genética , Reparo do DNA/genética , Medicina de Precisão , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Antineoplásicos/uso terapêutico , Proteínas Mutadas de Ataxia Telangiectasia/genética , Ensaios Clínicos como Assunto , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Genes BRCA1 , Genes BRCA2 , Mutação em Linhagem Germinativa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Indazóis/uso terapêutico , Indóis/uso terapêutico , Masculino , Ftalazinas/uso terapêutico , Piperazinas/uso terapêutico , Piperidinas/uso terapêutico , Compostos de Platina/uso terapêutico , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , PrognósticoRESUMO
The primary objective of this phase I study of LY2780301, a dual p70 S6 kinase and Akt inhibitor, was to determine the recommended phase II dose as a single agent in patients with advanced cancer. Secondary objectives included safety, pharmacokinetic, and pharmacodynamic analyses, and co-clinical analyses in Avatar models. Eligible patients received total daily doses of LY2780301 100-500 mg, given orally as a single dose or divided into 2 doses for 28-day cycles. Dose escalation followed 3 + 3 design. The primary pharmacodynamic endpoint was inhibition of S6 assessed by skin and tumor biopsy. Thirty-two patients were treated. Common toxicities possibly related to treatment included constipation (19 %), fatigue (13 %), nausea (9 %), and diarrhea (9 %). Grade 3/4 toxicities potentially related to treatment were anemia (n = 2), increased alanine aminotransferase/aspartate aminotransferase (ALT) (n = 1), and increased gamma-glutamyl transpeptidase (GGT) (n = 1). One patient experienced best overall response of prolonged stable disease for 6 cycles. Plasma exposures of LY2780301 exceeded predicted efficacious exposures, but were not dose proportional. Among patients receiving 500 mg daily >50 % exhibited reduced S6 in skin biopsies at Day 8 of treatment, but the effect was not maintained. Plasma concentrations of LY2780301 and/or its metabolites were not correlated with S6 expression in the epidermis. There was minimal antitumor activity against the model, CRC 019. Avatar models showed minimal pharmacodynamic effects consistent with the observed antitumor effects. This study suggests a dose of LY2780301 500 mg QD for future studies.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 70-kDa/antagonistas & inibidores , Adulto , Idoso , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Feminino , Humanos , Masculino , Camundongos Nus , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Neoplasias/enzimologia , Neoplasias/patologia , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto JovemRESUMO
Prostate cancer (PCa) is one of the leading causes of cancer-related deaths among men. Consequently, the identification of novel molecular targets for treatment is urgently needed to improve patients' outcomes. Our group recently reported that some elements of the cellular machinery controlling alternative-splicing might be useful as potential novel therapeutic tools against advanced PCa. However, the presence and functional role of RBM22, a key spliceosome component, in PCa remains unknown. Therefore, RBM22 levels were firstly interrogated in 3 human cohorts and 2 preclinical mouse models (TRAMP/Pbsn-Myc). Results were validated in in silico using 2 additional cohorts. Then, functional effects in response to RBM22 overexpression (proliferation, migration, tumorspheres/colonies formation) were tested in PCa models in vitro (LNCaP, 22Rv1, and PC-3 cell-lines) and in vivo (xenograft). High throughput methods (ie, RNA-seq, nCounter PanCancer Pathways Panel) were performed in RBM22 overexpressing cells and xenograft tumors. We found that RBM22 levels were down-regulated (mRNA and protein) in PCa samples, and were inversely associated with key clinical aggressiveness features. Consistently, a gradual reduction of RBM22 from non-tumor to poorly differentiated PCa samples was observed in transgenic models (TRAMP/Pbsn-Myc). Notably, RBM22 overexpression decreased aggressiveness features in vitro, and in vivo. These actions were associated with the splicing dysregulation of numerous genes and to the downregulation of critical upstream regulators of cell-cycle (i.e., CDK1/CCND1/EPAS1). Altogether, our data demonstrate that RBM22 plays a critical pathophysiological role in PCa and invites to suggest that targeting negative regulators of RBM22 expression/activity could represent a novel therapeutic strategy to tackle this disease.
Assuntos
Processamento Alternativo , Neoplasias da Próstata , Masculino , Humanos , Animais , Camundongos , Processamento Alternativo/genética , Neoplasias da Próstata/metabolismo , Splicing de RNA , Spliceossomos , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão GênicaRESUMO
BACKGROUND: Several studies have reported the association of germline BRCA2 (gBRCA2) mutations with poor clinical outcomes in prostate cancer (PCa), but the impact of concurrent somatic events on gBRCA2 carriers survival and disease progression is unknown. PATIENTS AND METHODS: To ascertain the role of frequent somatic genomic alterations and histology subtypes in the outcomes of gBRCA2 mutation carriers and non-carriers, we correlated the tumour characteristics and clinical outcomes of 73 gBRCA2 and 127 non-carriers. Fluorescent in-situ hybridisation and next-generation sequencing were used to detect copy number variations in BRCA2, RB1, MYC and PTEN. Presence of intraductal and cribriform subtypes was also assessed. The independent impact of these events on cause-specific survival (CSS), metastasis-free survival and time to castration-resistant disease was assessed using cox-regression models. RESULTS: Somatic BRCA2-RB1 co-deletion (41% versus 12%, p < 0.001) and MYC amplification (53.4% versus 18.8%, p < 0.001) were enriched in gBRCA2 compared to sporadic tumours. Median CSS from diagnosis of PCa was 9.1 versus 17.6 years in gBRCA2 carriers and non-carriers, respectively (HR 2.12; p = 0.002), Median CSS in gBRCA2 carriers increased to 11.3 and 13.4 years in the absence of BRCA2-RB1 deletion or MYC amplification, respectively. Median CSS of non-carriers decreased to 8 and 2.6 years if BRCA2-RB1 deletion or MYC amplification were detected. CONCLUSIONS: gBRCA2-related prostate tumours are enriched for aggressive genomic features, such as BRCA2-RB1 co-deletion and MYC amplification. The presence or absence of these events modify the outcomes of gBRCA2 carriers.
Assuntos
Variações do Número de Cópias de DNA , Neoplasias da Próstata , Masculino , Humanos , Neoplasias da Próstata/patologia , Proteína BRCA2/genética , Heterozigoto , Mutação , Células Germinativas/patologia , Mutação em Linhagem GerminativaRESUMO
New disease specific biomarkers, especially for cancer, are urgently needed to improve individual diagnosis, prognosis, and treatment selection, that is, for personalized medicine. Genetic mutations that affect protein function drive cancer. Therefore, the detection of such mutations represents a source of cancer specific biomarkers. Here we confirm the implementation of the mutant protein specific immuno-SRM (where SRM is selective reaction monitoring) mass spectrometry method of RAS proteins reported by Wang et al. [Proc. Natl. Acad. Sci. USA 2011, 108, 2444-2449], which exploits an antibody to simultaneously capture the different forms of the target protein and the resolving power and sensitivity of LC-MS/MS and improve the technique by using a more sensitive mass spectrometer. The mutant form G12D was quantified by SRM on a QTRAP 5500 mass spectrometer and the MIDAS workflow was used to confirm the sequence of the targeted peptides. This assay has been applied to quantify wild type and mutant RAS proteins in patient tumors, xenografted human tissue, and benign human epidermal tumors at high sensitivity. The limit of detection for the target proteins was as low as 12 amol (0.25 pg). It requires low starting amounts of tissue (ca.15 mg) that could be obtained from a needle aspiration biopsy. The described strategy could find application in the clinical arena and be applied to the study of expression of protein variants in disease.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias Colorretais/química , Espectrometria de Massas/métodos , Proteínas Mutantes/análise , Proteínas de Neoplasias/análise , Neoplasias Pancreáticas/química , Animais , Calibragem , Neoplasias Colorretais/genética , Feminino , Humanos , Imunoprecipitação , Modelos Lineares , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/genética , Peptídeos/análise , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise Serial de Tecidos , Proteínas ras/análise , Proteínas ras/genéticaRESUMO
Circulating tumor cell (CTC) enumeration and changes following treatment have been demonstrated to be superior to PSA response in determining mCRPC outcome in patients receiving AR signaling inhibitors but not taxanes. We carried out a pooled analysis of two prospective studies in mCRPC patients treated with docetaxel. CTCs were measured at baseline and 3-6 weeks post treatment initiation. Cox regression models were constructed to compare 6-month radiographical progression-free survival (rPFS), CTCs and PSA changes predicting outcome. Among the subjects, 80 and 52 patients had evaluable baseline and post-treatment CTC counts, respectively. A significant association of higher baseline CTC count with worse overall survival (OS), PFS and time to PSA progression (TTPP) was observed. While CTC response at 3-6 weeks (CTC conversion (from ≥5 to <5 CTCs), CTC30 (≥30% decline in CTC) or CTC0 (decline to 0 CTC)) and 6-month rPFS were significantly associated with OS (all p < 0.005), the association was not significant for PSA30 or PSA50 response. CTC and PSA response were discordant in over 50% of cases, with outcome driven by CTC response in these patients. The c-index values for OS were superior for early CTC changes compared to PSA response endpoints, and similar to 6-month rPFS. Early CTC declines were good predictors of improved outcomes in mCRPC patients treated with docetaxel in this small study, offering a superior and/or earlier estimation of docetaxel benefit in comparison to PSA or rPFS that merits further confirmation in larger studies.
RESUMO
BACKGROUND: Intraductal (IDC) and cribriform (CRIB) histologies in prostate cancer have been associated with germline BRCA2 (gBRCA2) mutations in small retrospective series, leading to the recommendation of genetic testing for patients with IDC in the primary tumour. PATIENTS AND METHODS: To examine the association of gBRCA2 mutations and other tumour molecular features with IDC and/or cribriform (CRIB) histologies, we conducted a case-control study in which primary prostate tumours from 58 gBRCA2 carriers were matched (1:2) by Gleason Grade Group and specimen type to 116 non-carriers. Presence/absence of IDC and CRIB morphologies was established by two expert uropathologists blinded to gBRCA2 status. Fluorescent in-situ hybridization (FISH) and next-generation sequencing (NGS) were used to detect BRCA2 alterations, PTEN deletions and TMPRSS2-ERG fusions. Chi-squared tests were used to compare the frequency of IDC and CRIB in gBRCA2 carriers and controls and to assess associations with other variables. Logistic regression models were constructed to identify independent factors associated with both histology patterns. RESULTS: No significant differences between gBRCA2 carriers and non-carriers were observed in the prevalence of IDC (36% gBRCA2 versus 50% non-carriers, p = 0.085) or CRIB (53% gBRCA2 versus 43% non-carriers p = 0.197) patterns. However, IDC histology was independently associated with bi-allelic BRCA2 alterations (OR 4.3, 95%CI 1.1-16.2) and PTEN homozygous loss (OR 5.2, 95%CI 2.1-13.1). CRIB morphology was also independently associated with bi-allelic BRCA2 alterations (OR 5.6, 95%CI 1.7-19.3). CONCLUSIONS: While we found no association between gBRCA2 mutations and IDC or CRIB histologies, bi-allelic BRCA2 loss in primary prostate tumours was significantly associated with both variant morphologies, independently of other clinical-pathologic factors.
Assuntos
Proteína BRCA2/genética , Biomarcadores Tumorais/genética , Mutação , Neoplasias da Próstata/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Análise Mutacional de DNA , Deleção de Genes , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , PTEN Fosfo-Hidrolase/genética , Fenótipo , Neoplasias da Próstata/patologia , Medição de Risco , Fatores de Risco , EspanhaRESUMO
PURPOSE: The tumor microenvironment plays a key role in cancer development and progression and is involved in resistance to chemo- and immunotherapy. Cancer-associated fibroblast expressing fibroblast-activating protein α (FAPα) is one of the predominant stroma cell types and is involved in resistance to immunotherapy. EXPERIMENTAL DESIGN: We generated OMTX705, a novel antibody-drug conjugate from a humanized anti-FAP antibody linked to a new cytolysin. Here, we studied its antineoplastic activity in vitro and in preclinical mouse models alone and in combination with chemotherapy as well as immunotherapy in PD-1-resistant tumors. RESULTS: In Avatar models, OMTX705 showed a 100% tumor growth inhibition and prolonged tumor regressions as single agent and in combination with chemotherapy. Treatment rechallenge following treatment discontinuation induced additional tumor regression, suggesting lack of treatment resistance. In a mouse model with a humanized immune system resistant to PD-1 inhibition, OMTX705 increased tumor infiltration by CD8+ T cells, induced complete regressions, and delayed tumor recurrence. CONCLUSIONS: These data suggest that FAP targeting with OMTX705 represents a novel and potent strategy for cancer treatment, including tumors resistant to immunotherapy, and support its clinical development.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Imunoconjugados/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Endopeptidases , Humanos , Imunomodulação/efeitos dos fármacos , Camundongos , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Inhibition of the cell-cycle kinases CDK4 and CDK6 is now part of the standard treatment in advanced breast cancer. CDK4/6 inhibitors, however, are not expected to cooperate with DNA-damaging or antimitotic chemotherapies as the former prevent cell-cycle entry, thus interfering with S-phase- or mitosis-targeting agents. Here, we report that sequential administration of CDK4/6 inhibitors after taxanes cooperates to prevent cellular proliferation in pancreatic ductal adenocarcinoma (PDAC) cells, patient-derived xenografts, and genetically engineered mice with Kras G12V and Cdkn2a-null mutations frequently observed in PDAC. This effect correlates with the repressive activity of CDK4/6 inhibitors on homologous recombination proteins required for the recovery from chromosomal damage. CDK4/6 inhibitors also prevent recovery from multiple DNA-damaging agents, suggesting broad applicability for their sequential administration after available chemotherapeutic agents.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Ductal Pancreático/tratamento farmacológico , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Neoplasias Pancreáticas/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Albuminas/administração & dosagem , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Inibidor p16 de Quinase Dependente de Ciclina/genética , Reparo do DNA/efeitos dos fármacos , Esquema de Medicação , Recombinação Homóloga/efeitos dos fármacos , Humanos , Camundongos Nus , Camundongos Transgênicos , Mutação , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Paclitaxel/administração & dosagem , Neoplasias Pancreáticas/patologia , Piperazinas/administração & dosagem , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas p21(ras)/genética , Piridinas/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Metastasis development is the leading cause of cancer-related mortality in pancreatic ductal adenocarcinoma (PDAC) and yet, few preclinical systems to recapitulate its full spreading process are available. Thus, modeling of tumor progression to metastasis is urgently needed. In this work, we describe the generation of highly metastatic PDAC patient-derived xenograft (PDX) mouse models and subsequent single-cell RNA-sequencing (RNA-seq) of circulating tumor cells (CTC), isolated by human HLA sorting, to identify altered signaling and metabolic pathways, as well as potential therapeutic targets. The mouse models developed liver and lung metastasis with a high reproducibility rate. Isolated CTCs were highly tumorigenic, had metastatic potential, and single-cell RNA-seq showed that their expression profiles clustered separately from those of their matched primary and metastatic tumors and were characterized by low expression of cell-cycle and extracellular matrix-associated genes. CTC transcriptomics identified survivin (BIRC5), a key regulator of mitosis and apoptosis, as one of the highest upregulated genes during metastatic spread. Pharmacologic inhibition of survivin with YM155 or survivin knockdown promoted cell death in organoid models as well as anoikis, suggesting that survivin facilitates cancer cell survival in circulation. Treatment of metastatic PDX models with YM155 alone and in combination with chemotherapy hindered the metastatic development resulting in improved survival. Metastatic PDX mouse model development allowed the identification of survivin as a promising therapeutic target to prevent the metastatic dissemination in PDAC.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/secundário , Células Neoplásicas Circulantes/patologia , Neoplasias Pancreáticas/patologia , Análise de Célula Única/métodos , Transcriptoma , Animais , Apoptose , Carcinoma Ductal Pancreático/sangue , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/genética , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Defects in tumor-intrinsic interferon (IFN) signaling result in failure of immune checkpoint blockade (ICB) against cancer, but these tumors may still maintain sensitivity to T cell-based adoptive cell therapy (ACT). We generated models of IFN signaling defects in B16 murine melanoma observed in patients with acquired resistance to ICB. Tumors lacking Jak1 or Jak2 did not respond to ICB, whereas ACT was effective against Jak2 KO tumors, but not Jak1 KO tumors, where both type I and II tumor IFN signaling were defective. This was a direct result of low baseline class I major histocompatibility complex (MHC I) expression in B16 and the dependency of MHC I expression on either type I or type II IFN signaling. We used genetic and pharmacologic approaches to uncouple this dependency and restore MHC I expression. Through independent mechanisms, overexpression of NLRC5 (nucleotide-binding oligomerization domain-like receptor family caspase recruitment domain containing 5) and intratumoral delivery of BO-112, a potent nanoplexed version of polyinosinic:polycytidylic acid (poly I:C), each restored the efficacy of ACT against B16-Jak1 KO tumors. BO-112 activated double-stranded RNA (dsRNA) sensing (via protein kinase R and Toll-like receptor 3) and induced MHC I expression via nuclear factor κB, independent of both IFN signaling and NLRC5. In summary, we demonstrated that in the absence of tumor IFN signaling, MHC I expression is essential and sufficient for the efficacy of ACT. For tumors lacking MHC I expression due to deficient IFN signaling, activation of dsRNA sensors by BO-112 affords an alternative approach to restore the efficacy of ACT.
Assuntos
Apresentação de Antígeno , Interferon gama , Animais , Humanos , Imunoterapia , Peptídeos e Proteínas de Sinalização Intracelular , Janus Quinase 1 , Camundongos , NF-kappa B , Transdução de SinaisRESUMO
Intratumoral therapies, especially Toll-like receptor agonists, can trigger both the innate and adaptive immune systems. BO-112 is a nanoplexed form of polyinosinic:polycytidylic acid (poly I:C) that induces local and systemic immunotherapeutic effects in mouse models. In a multicenter phase 1 clinical trial, repeated intratumoral administrations of BO-112 induced an increase in tumor cell necrosis and apoptosis, as well as augmented immune reactivity according to gene expression profiling. The first three cohorts receiving BO-112 as a monotherapy resulted in a recommended dose of 1 mg that could be safely repeated. Two grade 3 to 4 adverse reactions in the form of reversible thrombocytopenia were reported. In a fourth cohort of 28 patients with tumors that had primary resistance to anti-programmed cell death protein-1 (PD-1), the combination of intratumoral BO-112 with nivolumab or pembrolizumab was also well tolerated, and 3 patients (2 with melanoma and 1 with renal cell carcinoma) achieved partial responses, with 10 more patients having stable disease at 8 to 12 weeks. Thus, local BO-112 combined with a systemic anti-PD-1 agent might be a strategy to revert anti-PD-1 resistance.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Melanoma , Animais , Humanos , Melanoma/tratamento farmacológico , Camundongos , Nivolumabe/uso terapêutico , Poli IRESUMO
Different RNA interference (RNAi) components participate in post-transcriptional regulation via RNA silencing. The expression pattern of the genes Drosha and Dicer and the members of the Argonaute family Ago1, Ago2, Ago3 and Ago4, all elements participating in the RNAi pathways, were investigated in mouse somatic tissues and testis using quantitative RT-PCR. Expression patterns of different testis cells and those emerging during testis development were also investigated. The differential patterns of expression seen suggest potential pleiotropic roles for certain components of the RNAi machinery. Both spermatocytes and spermatids showed a defined gene expression pattern. The strong expression of Ago4 in germ cells suggests that this protein plays a key role in germ-cell differentiation in the seminiferous epithelium.
Assuntos
Interferência de RNA , Complexo de Inativação Induzido por RNA/metabolismo , Ribonuclease III/metabolismo , Testículo/metabolismo , Animais , Expressão Gênica , Masculino , Camundongos , RNA Mensageiro/metabolismo , Complexo de Inativação Induzido por RNA/genética , Ribonuclease III/genética , Espermatogênese/genética , Distribuição TecidualRESUMO
So far, the success of anticancer nanomedicines has been moderate due to their lack of adequate targeting properties and/or to their difficulties for penetrating tumors. Here we report a multifunctional drug nanocarrier consisting of hyaluronic acid nanocapsules conjugated with the tumor homing peptide tLyp1, which exhibits both, dual targeting properties (to the tumor and to the lymphatics), and enhanced tumor penetration. Data from a 3D co-culture in vitro model showed the capacity of these nanocapsules to interact with the NRP1 receptors over-expressed in cancer cells. The targeting capacity of the nanocapsules was evidenced in orthotopic lung cancer-bearing mice, using docetaxel as a standard drug. The results showed a dramatic accumulation of docetaxel in the tumor (37-fold the one achieved with Taxotere®). This biodistribution profile correlated with the high efficacy shown in terms of tumor growth regression and drastic reduction of metastasis in the lymphatics. When efficacy was validated in a pancreatic patient-derived tumor, the nanocapsule's activity was comparable to that of a dose ten times higher of Abraxane®. Multi-functionality was found to be the key to the success of this new therapy.
Assuntos
Antineoplásicos/administração & dosagem , Docetaxel/administração & dosagem , Portadores de Fármacos/administração & dosagem , Ácido Hialurônico/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Células A549 , Animais , Antineoplásicos/farmacocinética , Técnicas de Cocultura , Docetaxel/farmacocinética , Portadores de Fármacos/farmacocinética , Feminino , Humanos , Ácido Hialurônico/farmacocinética , Células Jurkat , Neoplasias Pulmonares/metabolismo , Camundongos Nus , Distribuição TecidualRESUMO
BACKGROUND: RNA interference (RNAi) is a valuable tool in the investigation of gene function. The purpose of this study was to examine the availability, target cell types and efficiency of RNAi in the mouse seminiferous epithelium. METHODS: The experimental model was based on transgenic mice expressing EGFP (enhanced green fluorescent protein). RNAi was induced by in vivo transfection of plasmid vectors encoding for short hairpin RNAs (shRNAs) targeting EGFP. shRNAs were transfected in vivo by microinjection into the seminiferous tubules via the rete testis followed by square wave electroporation. As a transfection reporter, expression of red fluorescent protein (HcRed 1) was used. Cell types, the efficiency of both transfections and RNAi were all evaluated. RESULTS: Sertoli cells were the main transfected cells. A reduction of about 40% in the level of EGFP protein was detected in cells successfully transfected both in vivo and in vitro. However, the efficiency of in vivo transfection was low. CONCLUSION: In adult seminiferous epithelial cells, in vivo post-transcriptional gene silencing mediated by RNAi via shRNA is efficient in Sertoli cells. Similar levels of RNAi were detected both in vivo and in vitro. This also indicates that Sertoli cells have the necessary silencing machinery to repress the expression of endogenous genes via RNAi.
Assuntos
Inativação Gênica , Interferência de RNA , Epitélio Seminífero/fisiologia , Células de Sertoli/fisiologia , Animais , Células Cultivadas , Eletroporação , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Proteínas de Fluorescência Verde/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Epitélio Seminífero/citologia , Células de Sertoli/citologia , TransfecçãoRESUMO
In the present work we report the synthesis of four new ER ligands which can be used as scaffolds for the introduction of the basic side chains necessary for antiestrogenic activity. Affinities and agonist/antagonist characterization of the ligands for both ERalpha and ERbeta have been determined in a competitive radioligand assay, and in an in vitro coactivator recruitment functional assay, respectively. Molecular modelling techniques have been used in order to rationalize the experimental results. Compound is reported as a novel ERbeta-agonist/ERalpha-antagonist. Two compounds show an interesting antitumour profile towards two pancreatic cancer cell lines and have been selected for in vivo assays.
Assuntos
Desenho de Fármacos , Moduladores Seletivos de Receptor Estrogênico/síntese química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Estradiol/metabolismo , Receptor alfa de Estrogênio/agonistas , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor alfa de Estrogênio/química , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/agonistas , Receptor beta de Estrogênio/antagonistas & inibidores , Receptor beta de Estrogênio/química , Receptor beta de Estrogênio/metabolismo , Humanos , Modelos Moleculares , Conformação Molecular , Moduladores Seletivos de Receptor Estrogênico/química , Moduladores Seletivos de Receptor Estrogênico/metabolismo , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Especificidade por SubstratoRESUMO
Purpose: Since drug responses vary between patients, it is crucial to develop pre-clinical or co-clinical strategies that forecast patient response. In this study, we tested whether RNA-based therapeutics were suitable for personalized medicine by using patient-derived-organoid (PDO) and patient-derived-xenograft (PDX) models.Experimental Design: We performed microRNA (miRNA) profiling of PDX samples to determine the status of miRNA deregulation in individual pancreatic ductal adenocarcinoma (PDAC) patients. To deliver personalized RNA-based-therapy targeting oncogenic miRNAs that form part of this common PDAC miRNA over-expression signature, we packaged antimiR oligonucleotides against one of these miRNAs in tumor-penetrating nanocomplexes (TPN) targeting cell surface proteins on PDAC tumors.Results: As a validation for our pre-clinical strategy, the therapeutic potential of one of our nano-drugs, TPN-21, was first shown to decrease tumor cell growth and survival in PDO avatars for individual patients, then in their PDX avatars.Conclusions: This general approach appears suitable for co-clinical validation of personalized RNA medicine and paves the way to prospectively identify patients with eligible miRNA profiles for personalized RNA-based therapy. Clin Cancer Res; 24(7); 1734-47. ©2018 AACR.
Assuntos
MicroRNAs/genética , Neoplasias Pancreáticas/genética , Animais , Carcinoma Ductal Pancreático/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Modelos Animais de Doenças , Perfilação da Expressão Gênica/métodos , Humanos , Camundongos , Camundongos Nus , Oncogenes/genética , Medicina de Precisão/métodos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Neoplasias PancreáticasRESUMO
A quarter of all solid tumors harbor KRAS oncogenes. Yet, no selective drugs have been approved to treat these malignancies. Genetic interrogation of the MAPK pathway revealed that systemic ablation of MEK or ERK kinases in adult mice prevent tumor development but are unacceptably toxic. Here, we demonstrate that ablation of c-RAF expression in advanced tumors driven by KrasG12V/Trp53 mutations leads to significant tumor regression with no detectable appearance of resistance mechanisms. Tumor regression results from massive apoptosis. Importantly, systemic abrogation of c-RAF expression does not inhibit canonical MAPK signaling, hence, resulting in limited toxicities. These results are of significant relevance for the design of therapeutic strategies to treat K-RAS mutant cancers.
Assuntos
Adenocarcinoma de Pulmão/genética , Genes ras/genética , Mutação/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas ras/genética , Animais , Linhagem Celular Tumoral , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas B-raf/genéticaRESUMO
On the basis of clinical trials using first-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), it became a doctrine that V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-RAS) mutations drive resistance to EGFR inhibition in non-small cell lung cancer (NSCLC). Conversely, we provide evidence that EGFR signaling is engaged in K-RAS-driven lung tumorigenesis in humans and in mice. Specifically, genetic mouse models revealed that deletion of Egfr quenches mutant K-RAS activity and transiently reduces tumor growth. However, EGFR inhibition initiates a rapid resistance mechanism involving non-EGFR ERBB family members. This tumor escape mechanism clarifies the disappointing outcome of first-generation TKIs and suggests high therapeutic potential of pan-ERBB inhibitors. On the basis of various experimental models including genetically engineered mouse models, patient-derived and cell line-derived xenografts, and in vitro experiments, we demonstrate that the U.S. Food and Drug Administration-approved pan-ERBB inhibitor afatinib effectively impairs K-RAS-driven lung tumorigenesis. Our data support reconsidering the use of pan-ERBB inhibition in clinical trials to treat K-RAS-mutated NSCLC.
Assuntos
Afatinib/uso terapêutico , Carcinogênese/patologia , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Proto-Oncogênicas p21(ras)/genética , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Afatinib/farmacologia , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/metabolismo , Cloridrato de Erlotinib/farmacologia , Cloridrato de Erlotinib/uso terapêutico , Gefitinibe/farmacologia , Gefitinibe/uso terapêutico , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mutação/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
In colorectal carcinoma patients, distant metastatic disease is present at initial diagnosis in nearly 25% of them. The majority of patients with metastatic colorectal carcinoma have incurable disease; therefore, new therapies are needed. Agents derived from medicinal plants have already demonstrated therapeutic activities in human cancer cells. Antartina is an antitumor agent isolated from Deschampsia antarctica Desv. This study aimed to evaluate the antitumor properties of Antartina in colorectal carcinoma models. We used human and murine colorectal carcinoma cell lines for investigating proliferation, apoptosis, and cell-cycle effects of Antartina therapy in vitro Avatar and immunocompetent colorectal carcinoma animal models were applied for evaluating the effects of Antartina in vivo Immune response against colorectal carcinoma model was investigated using CTL assay, analyzing dendritic cell activation and intratumor T-cell subpopulation, and by tumor rechallenge experiments. Antartina inhibits in vitro human colorectal carcinoma cell proliferation; however, in vivo experiments in Avatar colorectal carcinoma model Antartina display a limited antitumor effect. In an immunocompetent colorectal carcinoma mice model, Antartina potently inhibited tumor growth and liver metastases, leading to complete tumor regressions in >30% of mice and increased animal survival. In addition, Antartina induced a potent specific cytotoxic T-cell response against colorectal carcinoma and a long-lasting antitumor immunity. Interestingly, Antartina increased tumor immunogenicity and stimulated dendritic cell activation. No toxic effects were observed at the doses employed. Our findings showed that Antartina has the ability to induce antitumor immunity against colorectal carcinoma and can be used to develop new tools for the treatment of colorectal carcinoma. Mol Cancer Ther; 17(5); 966-76. ©2018 AACR.