RESUMEN
WRN helicase is a promising target for treatment of cancers with microsatellite instability (MSI) due to its essential role in resolving deleterious non-canonical DNA structures that accumulate in cells with faulty mismatch repair mechanisms1-5. Currently there are no approved drugs directly targeting human DNA or RNA helicases, in part owing to the challenging nature of developing potent and selective compounds to this class of proteins. Here we describe the chemoproteomics-enabled discovery of a clinical-stage, covalent allosteric inhibitor of WRN, VVD-133214. This compound selectively engages a cysteine (C727) located in a region of the helicase domain subject to interdomain movement during DNA unwinding. VVD-133214 binds WRN protein cooperatively with nucleotide and stabilizes compact conformations lacking the dynamic flexibility necessary for proper helicase function, resulting in widespread double-stranded DNA breaks, nuclear swelling and cell death in MSI-high (MSI-H), but not in microsatellite-stable, cells. The compound was well tolerated in mice and led to robust tumour regression in multiple MSI-H colorectal cancer cell lines and patient-derived xenograft models. Our work shows an allosteric approach for inhibition of WRN function that circumvents competition from an endogenous ATP cofactor in cancer cells, and designates VVD-133214 as a promising drug candidate for patients with MSI-H cancers.
Asunto(s)
Regulación Alostérica , Descubrimiento de Drogas , Inhibidores Enzimáticos , Proteómica , Helicasa del Síndrome de Werner , Animales , Femenino , Humanos , Masculino , Ratones , Regulación Alostérica/efectos de los fármacos , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/patología , Cisteína/efectos de los fármacos , Cisteína/metabolismo , Roturas del ADN de Doble Cadena/efectos de los fármacos , Descubrimiento de Drogas/métodos , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Inestabilidad de Microsatélites , Modelos Moleculares , Helicasa del Síndrome de Werner/antagonistas & inhibidores , Helicasa del Síndrome de Werner/química , Helicasa del Síndrome de Werner/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Muerte Celular/efectos de los fármacos , Adenosina Trifosfato/metabolismoRESUMEN
The Werner Syndrome helicase, WRN, is a promising therapeutic target in cancers with microsatellite instability (MSI). Long-term MSI leads to the expansion of TA nucleotide repeats proposed to form cruciform DNA structures, which in turn cause DNA breaks and cell lethality upon WRN downregulation. Here we employed biochemical assays to show that WRN helicase can efficiently and directly unfold cruciform structures, thereby preventing their cleavage by the SLX1-SLX4 structure-specific endonuclease. TA repeats are particularly prone to form cruciform structures, explaining why these DNA sequences are preferentially broken in MSI cells upon WRN downregulation. We further demonstrate that the activity of the DNA mismatch repair (MMR) complexes MutSα (MSH2-MSH6), MutSß (MSH2-MSH3), and MutLα (MLH1-PMS2) similarly decreases the level of DNA cruciforms, although the mechanism is different from that employed by WRN. When combined, WRN and MutLα exhibited higher than additive effects in in vitro cruciform processing, suggesting that WRN and the MMR proteins may cooperate. Our data explain how WRN and MMR defects cause genome instability in MSI cells with expanded TA repeats, and provide a mechanistic basis for their recently discovered synthetic-lethal interaction with promising applications in precision cancer therapy.
Asunto(s)
Reparación de la Incompatibilidad de ADN , ADN Cruciforme , Humanos , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Inestabilidad de Microsatélites , Helicasa del Síndrome de Werner/genética , Helicasa del Síndrome de Werner/metabolismo , Homólogo 1 de la Proteína MutL/genéticaRESUMEN
The therapeutic benefit of approved BRAF and MEK inhibitors (BRAFi/MEKi) in patients with brain metastatic BRAF V600E/K-mutated melanoma is limited and transient. Resistance largely occurs through the restoration of MAPK signaling via paradoxical BRAF activation, highlighting the need for more effective therapeutic options. Aiming to address this clinical challenge, we characterized the activity of a potent, brain-penetrant paradox breaker BRAFi (compound 1a, C1a) as first-line therapy and following progression upon treatment with approved BRAFi and BRAFi/MEKi therapies. C1a activity was evaluated in vitro and in vivo in melanoma cell lines and patient-derived models of BRAF V600E-mutant melanoma brain metastases following relapse after treatment with BRAFi/MEKi. C1a showed superior efficacy compared with approved BRAFi in both subcutaneous and brain metastatic models. Importantly, C1a manifested potent and prolonged antitumor activity even in models that progressed on BRAFi/MEKi treatment. Analysis of mechanisms of resistance to C1a revealed MAPK reactivation under drug treatment as the predominant resistance-driving event in both subcutaneous and intracranial tumors. Specifically, BRAF kinase domain duplication was identified as a frequently occurring driver of resistance to C1a. Combination therapies of C1a and anti-PD-1 antibody proved to significantly reduce disease recurrence. Collectively, these preclinical studies validate the outstanding antitumor activity of C1a in brain metastasis, support clinical investigation of this agent in patients pretreated with BRAFi/MEKi, unveil genetic drivers of tumor escape from C1a, and identify a combinatorial treatment that achieves long-lasting responses. SIGNIFICANCE: A brain-penetrant BRAF inhibitor demonstrates potent activity in brain metastatic melanoma, even upon relapse following standard BRAF inhibitor therapy, supporting further investigation into its clinical utility.
Asunto(s)
Neoplasias Encefálicas , Melanoma , Encéfalo/patología , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/patología , Quinasas de Proteína Quinasa Activadas por Mitógenos , Mutación , Recurrencia Local de Neoplasia/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-rafRESUMEN
MAPK inhibitors (MAPKi) remain an important component of the standard of care for metastatic melanoma. However, acquired resistance to these drugs limits their therapeutic benefit. Tumor cells can become refractory to MAPKi by reactivation of ERK. When this happens, tumors often become sensitive to drug withdrawal. This drug addiction phenotype results from the hyperactivation of the oncogenic pathway, a phenomenon commonly referred to as oncogene overdose. Several feedback mechanisms are involved in regulating ERK signaling. However, the genes that serve as gatekeepers of oncogene overdose in mutant melanoma remain unknown. Here, we demonstrate that depletion of the ERK phosphatase, DUSP4, leads to toxic levels of MAPK activation in both drug-naive and drug-resistant mutant melanoma cells. Importantly, ERK hyperactivation is associated with down-regulation of lineage-defining genes including MITF Our results offer an alternative therapeutic strategy to treat mutant melanoma patients with acquired MAPKi resistance and those unable to tolerate MAPKi.
Asunto(s)
Melanoma , Proteínas Proto-Oncogénicas B-raf , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Fosfatasas de Especificidad Dual/genética , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Humanos , Melanoma/genética , Melanoma/patología , Proteínas de la Membrana/metabolismo , Factor de Transcripción Asociado a Microftalmía/genética , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/genética , Oncogenes , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/genéticaRESUMEN
PURPOSE: Disease progression in BRAF V600E/K positive melanomas to approved BRAF/MEK inhibitor therapies is associated with the development of resistance mediated by RAF dimer inducing mechanisms. Moreover, progressing disease after BRAFi/MEKi frequently involves brain metastasis. Here we present the development of a novel BRAF inhibitor (Compound Ia) designed to address the limitations of available BRAFi/MEKi. EXPERIMENTAL DESIGN: The novel, brain penetrant, paradox breaker BRAFi is comprehensively characterized in vitro, ex vivo, and in several preclinical in vivo models of melanoma mimicking peripheral disease, brain metastatic disease, and acquired resistance to first-generation BRAFi. RESULTS: Compound Ia manifested elevated potency and selectivity, which triggered cytotoxic activity restricted to BRAF-mutated models and did not induce RAF paradoxical activation. In comparison to approved BRAFi at clinical relevant doses, this novel agent showed a substantially improved activity in a number of diverse BRAF V600E models. In addition, as a single agent, it outperformed a currently approved BRAFi/MEKi combination in a model of acquired resistance to clinically available BRAFi. Compound Ia presents high central nervous system (CNS) penetration and triggered evident superiority over approved BRAFi in a macro-metastatic and in a disseminated micro-metastatic brain model. Potent inhibition of MAPK by Compound Ia was also demonstrated in patient-derived tumor samples. CONCLUSIONS: The novel BRAFi demonstrates preclinically the potential to outperform available targeted therapies for the treatment of BRAF-mutant tumors, thus supporting its clinical investigation.
Asunto(s)
Melanoma , Proteínas Proto-Oncogénicas B-raf , Encéfalo/patología , Línea Celular Tumoral , Resistencia a Antineoplásicos , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/patología , Terapia Molecular Dirigida , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
Glioblastoma (GBM) is highly resistant to chemotherapies, immune-based therapies, and targeted inhibitors. To identify novel drug targets, we screened orthotopically implanted, patient-derived glioblastoma sphere-forming cells using an RNAi library to probe essential tumor cell metabolic programs. This identified high dependence on mitochondrial fatty acid metabolism. We focused on medium-chain acyl-CoA dehydrogenase (MCAD), which oxidizes medium-chain fatty acids (MCFA), due to its consistently high score and high expression among models and upregulation in GBM compared with normal brain. Beyond the expected energetics impairment, MCAD depletion in primary GBM models induced an irreversible cascade of detrimental metabolic effects characterized by accumulation of unmetabolized MCFAs, which induced lipid peroxidation and oxidative stress, irreversible mitochondrial damage, and apoptosis. Our data uncover a novel protective role for MCAD to clear lipid molecules that may cause lethal cell damage, suggesting that therapeutic targeting of MCFA catabolism may exploit a key metabolic feature of GBM. SIGNIFICANCE: MCAD exerts a protective role to prevent accumulation of toxic metabolic by-products in glioma cells, actively catabolizing lipid species that would otherwise affect mitochondrial integrity and induce cell death. This work represents a first demonstration of a nonenergetic role for dependence on fatty acid metabolism in cancer.This article is highlighted in the In This Issue feature, p. 2659.
Asunto(s)
Acil-CoA Deshidrogenasa , Glioblastoma , Peroxidación de Lípido , Mitocondrias , Acil-CoA Deshidrogenasa/metabolismo , Apoptosis , Ácidos Grasos/metabolismo , Glioblastoma/enzimología , Glioblastoma/genética , Humanos , Mitocondrias/metabolismo , Estrés OxidativoRESUMEN
[This corrects the article DOI: 10.1155/2014/895986.].
RESUMEN
Proteolysis targeting chimeras are bifunctional small molecules capable of recruiting a target protein of interest to an E3 ubiquitin ligase that facilitates target ubiquitination followed by proteasome-mediated degradation. The first molecules acting on this novel therapeutic paradigm have just entered clinical testing. Here, by using Bromodomain Containing 4 (BRD4) degraders engaging cereblon and Von Hippel-Lindau E3 ligases, we investigated key determinants of resistance to this new mode of action. A loss-of-function screen for genes required for BRD4 degradation revealed strong dependence on the E2 and E3 ubiquitin ligases as well as for members of the COP9 signalosome complex for both cereblon- and Von Hippel-Lindau-engaging BRD4 degraders. Cancer cell lines raised to resist BRD4 degraders manifested a degrader-specific mechanism of resistance, resulting from the loss of components of the ubiquitin proteasome system. In addition, degrader profiling in a cancer cell line panel revealed a differential pattern of activity of Von Hippel-Lindau- and cereblon-based degraders, highlighting the need for the identification of degradation-predictive biomarkers enabling effective patient stratification.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Azepinas/farmacología , Proteínas de Ciclo Celular/metabolismo , Resistencia a Medicamentos/efectos de los fármacos , Factores de Transcripción/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Proteínas de Ciclo Celular/química , Línea Celular Tumoral , Dipéptidos/farmacología , Células HEK293 , Humanos , Ftalimidas/farmacología , Prueba de Estudio Conceptual , Proteolisis , Factores de Transcripción/química , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) remains recalcitrant to all forms of cancer treatment and carries a five-year survival rate of only 8%1. Inhibition of oncogenic KRAS (hereafter KRAS*), the earliest lesion in disease development that is present in more than 90% of PDACs, and its signalling surrogates has yielded encouraging preclinical results with experimental agents2-4. However, KRAS*-independent disease recurrence following genetic extinction of Kras* in mouse models anticipates the need for co-extinction strategies5,6. Multiple oncogenic processes are initiated at the cell surface, where KRAS* physically and functionally interacts to direct signalling that is essential for malignant transformation and tumour maintenance. Insights into the complexity of the functional cell-surface-protein repertoire (surfaceome) have been technologically limited until recently and-in the case of PDAC-the genetic control of the function and composition of the PDAC surfaceome in the context of KRAS* signalling remains largely unknown. Here we develop an unbiased, functional target-discovery platform to query KRAS*-dependent changes of the PDAC surfaceome, which reveals syndecan 1 (SDC1, also known as CD138) as a protein that is upregulated at the cell surface by KRAS*. Localization of SDC1 at the cell surface-where it regulates macropinocytosis, an essential metabolic pathway that fuels PDAC cell growth-is essential for disease maintenance and progression. Thus, our study forges a mechanistic link between KRAS* signalling and a targetable molecule driving nutrient salvage pathways in PDAC and validates oncogene-driven surfaceome annotation as a strategy to identify cancer-specific vulnerabilities.
Asunto(s)
Carcinoma Ductal Pancreático/patología , Neoplasias Pancreáticas/patología , Pinocitosis , Sindecano-1/metabolismo , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/metabolismo , Animales , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Proliferación Celular , Progresión de la Enfermedad , Femenino , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Masculino , Ratones , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Transducción de SeñalRESUMEN
Acquired and intrinsic resistance to receptor tyrosine kinase inhibitors (RTKi) represents a major hurdle in improving the management of clear cell renal cell carcinoma (ccRCC). Recent reports suggest that drug resistance is driven by tumor adaptation via epigenetic mechanisms that activate alternative survival pathways. The histone methyl transferase EZH2 is frequently altered in many cancers, including ccRCC. To evaluate its role in ccRCC resistance to RTKi, we established and characterized a spontaneously metastatic, patient-derived xenograft model that is intrinsically resistant to the RTKi sunitinib, but not to the VEGF therapeutic antibody bevacizumab. Sunitinib maintained its antiangiogenic and antimetastatic activity but lost its direct antitumor effects due to kinome reprogramming, which resulted in suppression of proapoptotic and cell-cycle-regulatory target genes. Modulating EZH2 expression or activity suppressed phosphorylation of certain RTKs, restoring the antitumor effects of sunitinib in models of acquired or intrinsically resistant ccRCC. Overall, our results highlight EZH2 as a rational target for therapeutic intervention in sunitinib-resistant ccRCC as well as a predictive marker for RTKi response in this disease. Cancer Res; 77(23); 6651-66. ©2017 AACR.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Células Renales/tratamiento farmacológico , Resistencia a Antineoplásicos/fisiología , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Indoles/farmacología , Neoplasias Renales/tratamiento farmacológico , Pirroles/farmacología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Animales , Bevacizumab/farmacología , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Proteína Potenciadora del Homólogo Zeste 2/genética , Femenino , Humanos , Neoplasias Renales/patología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/secundario , Ratones , Ratones Endogámicos ICR , Ratones SCID , Neovascularización Patológica/tratamiento farmacológico , Fosforilación , Proteínas Tirosina Quinasas Receptoras/metabolismo , Sunitinib , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Malignant neoplasms evolve in response to changes in oncogenic signalling. Cancer cell plasticity in response to evolutionary pressures is fundamental to tumour progression and the development of therapeutic resistance. Here we determine the molecular and cellular mechanisms of cancer cell plasticity in a conditional oncogenic Kras mouse model of pancreatic ductal adenocarcinoma (PDAC), a malignancy that displays considerable phenotypic diversity and morphological heterogeneity. In this model, stochastic extinction of oncogenic Kras signalling and emergence of Kras-independent escaper populations (cells that acquire oncogenic properties) are associated with de-differentiation and aggressive biological behaviour. Transcriptomic and functional analyses of Kras-independent escapers reveal the presence of Smarcb1-Myc-network-driven mesenchymal reprogramming and independence from MAPK signalling. A somatic mosaic model of PDAC, which allows time-restricted perturbation of cell fate, shows that depletion of Smarcb1 activates the Myc network, driving an anabolic switch that increases protein metabolism and adaptive activation of endoplasmic-reticulum-stress-induced survival pathways. Increased protein turnover renders mesenchymal sub-populations highly susceptible to pharmacological and genetic perturbation of the cellular proteostatic machinery and the IRE1-α-MKK4 arm of the endoplasmic-reticulum-stress-response pathway. Specifically, combination regimens that impair the unfolded protein responses block the emergence of aggressive mesenchymal subpopulations in mouse and patient-derived PDAC models. These molecular and biological insights inform a potential therapeutic strategy for targeting aggressive mesenchymal features of PDAC.
Asunto(s)
Mesodermo/patología , Neoplasias Pancreáticas/patología , Animales , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Estrés del Retículo Endoplásmico/genética , Femenino , Genes myc , Genes ras , Humanos , MAP Quinasa Quinasa 4/metabolismo , Sistema de Señalización de MAP Quinasas , Masculino , Mesodermo/metabolismo , Ratones , Mosaicismo , Proteína Oncogénica p55(v-myc)/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Proteolisis , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteína SMARCB1/deficiencia , Proteína SMARCB1/metabolismo , Transcriptoma/genética , GemcitabinaRESUMEN
Current treatment regimens for pancreatic ductal adenocarcinoma (PDAC) yield poor 5-year survival, emphasizing the critical need to identify druggable targets essential for PDAC maintenance. We developed an unbiased and in vivo target discovery approach to identify molecular vulnerabilities in low-passage and patient-derived PDAC xenografts or genetically engineered mouse model-derived allografts. Focusing on epigenetic regulators, we identified WDR5, a core member of the COMPASS histone H3 Lys4 (H3K4) MLL (1-4) methyltransferase complex, as a top tumor maintenance hit required across multiple human and mouse tumors. Mechanistically, WDR5 functions to sustain proper execution of DNA replication in PDAC cells, as previously suggested by replication stress studies involving MLL1, and c-Myc, also found to interact with WDR5. We indeed demonstrate that interaction with c-Myc is critical for this function. By showing that ATR inhibition mimicked the effects of WDR5 suppression, these data provide rationale to test ATR and WDR5 inhibitors for activity in this disease.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Carcinogénesis/metabolismo , Carcinogénesis/patología , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Proliferación Celular , Daño del ADN , Progresión de la Enfermedad , Epigénesis Genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Lentivirus/metabolismo , Ratones , Modelos Biológicos , Complejos Multiproteicos/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Unión Proteica , Subunidades de Proteína/metabolismo , ARN Interferente Pequeño/metabolismo , Estrés FisiológicoRESUMEN
Several reports indicate that chemo-resistant cancer cells become highly adapted to intrinsic oxidative stress by up-regulating their antioxidant systems, which causes an increase of intracellular GSH content. Doxorubicin is one of the most widely used drugs for tumor treatment, able to kill cancer cells through several mechanisms. However, doxorubicin use is limited by its toxicity and cancer resistance. Therefore, new therapeutic strategies able to reduce doses and to overcome chemo-resistance are needed. A new class of glutathione-responsive cyclodextrin nanosponges (GSH-NS), is able to release anticancer drugs preferentially in cells having high GSH content. Doxorubicin-loaded GSH-NS, in the cancer cells with high GSH content, inhibited clonogenic growth, cell viability, topoisomerase II activity and induced DNA damage with higher effectiveness than free drug. Moreover, GSH-NS reduced the development of human tumor in xenograft models more than free drug. These characteristics indicate that GSH-NS can be a suitable drug delivery carrier for future applications in cancer therapy.
Asunto(s)
Sistemas de Liberación de Medicamentos , Resistencia a Antineoplásicos/genética , Neoplasias/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Animales , Antioxidantes/química , Antioxidantes/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Daño del ADN/efectos de los fármacos , Doxorrubicina/administración & dosificación , Doxorrubicina/química , Glutatión/química , Glutatión/metabolismo , Humanos , Ratones , Nanoestructuras/administración & dosificación , Nanoestructuras/química , Neoplasias/metabolismo , Neoplasias/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Despite marked tumor shrinkage after 5-FU treatment, the frequency of colon cancer relapse indicates that a fraction of tumor cells survives treatment causing tumor recurrence. The majority of cancer cells divert metabolites into anabolic pathways through Warburg behavior giving an advantage in terms of tumor growth. Here, we report that treatment of colon cancer cell with 5-FU selects for cells with mesenchymal stem-like properties that undergo a metabolic reprogramming resulting in addiction to OXPHOS to meet energy demands. 5-FU treatment-resistant cells show a de novo expression of pyruvate kinase M1 (PKM1) and repression of PKM2, correlating with repression of the pentose phosphate pathway, decrease in NADPH level and in antioxidant defenses, promoting PKM2 oxidation and acquisition of stem-like phenotype. Response to 5-FU in a xenotransplantation model of human colon cancer confirms activation of mitochondrial function. Combined treatment with 5-FU and a pharmacological inhibitor of OXPHOS abolished the spherogenic potential of colon cancer cells and diminished the expression of stem-like markers. These findings suggest that inhibition of OXPHOS in combination with 5-FU is a rational combination strategy to achieve durable treatment response in colon cancer.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Neoplasias del Colon/tratamiento farmacológico , Resistencia a Antineoplásicos , Fluorouracilo/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Fosforilación Oxidativa , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Antioxidantes/metabolismo , Biomarcadores de Tumor/metabolismo , Proteínas Portadoras/metabolismo , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Inhibidores Enzimáticos/farmacología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Femenino , Células HT29 , Humanos , Proteínas de la Membrana/metabolismo , Ratones Desnudos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , NADP/metabolismo , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Oxidación-Reducción , Fosforilación Oxidativa/efectos de los fármacos , Fenotipo , Piruvato Quinasa/metabolismo , Hormonas Tiroideas/metabolismo , Factores de Tiempo , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas de Unión a Hormona TiroideRESUMEN
4-hydroxynonenal (HNE), a lipid peroxidation product, is a promising anti-neoplastic drug due to its remarkable anti-cancer activities. However, this possibility has not been explored, because the delivery of HNE is very challenging as a result of its low solubility and its poor stability. This study intentionally designed a new type of lipid nanocapsules specifically for HNE delivery. They consist of a medium chain triglyceride liquid oil core surrounded by a polymer shell. A ß-cyclodextrin-poly(4-acryloylmorpholine) conjugate was selected as the shell component. HNE-loaded nanocapsules were about 350 nm in size with a negative surface charge. They were stable for two years when stored in suspensions at 4 degrees C. In vitro experiments showed that HNE was released from the nanocapsules at a considerable rate. Nanocapsule uptake into cells was evaluated using a fluorescent formulation that revealed rapid internalisation. Cytotoxicity studies demonstrated the safety of the formulation. Enhanced anti-tumoral activity against various cell lines, depending on increased HNE stability, was obtained by using HNE-loaded nanocapsules. In particular, we have demonstrated an increase in anti-proliferative, pro-apoptotic and differentiative activity in several tumour cell lines from different tissues. Moreover, we evaluated the effects of these new nanocapsules on a three-dimensional human reconstructed model of skin melanoma. Interestingly, the encouraging results obtained with topical administration on the epidermal surface could open new perspectives in melanoma treatments.
Asunto(s)
Aldehídos/química , Aldehídos/farmacología , Portadores de Fármacos/química , Lípidos/química , Melanoma/patología , Nanocápsulas/química , Acrilamidas/química , Transporte Biológico , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ciclodextrinas/química , Estabilidad de Medicamentos , Humanos , Morfolinas/químicaRESUMEN
Myelodysplastic syndrome (MDS) risk correlates with advancing age, therapy-induced DNA damage, and/or shorter telomeres, but whether telomere erosion directly induces MDS is unknown. Here, we provide the genetic evidence that telomere dysfunction-induced DNA damage drives classical MDS phenotypes and alters common myeloid progenitor (CMP) differentiation by repressing the expression of mRNA splicing/processing genes, including SRSF2. RNA-seq analyses of telomere dysfunctional CMP identified aberrantly spliced transcripts linked to pathways relevant to MDS pathogenesis such as genome stability, DNA repair, chromatin remodeling, and histone modification, which are also enriched in mouse CMP haploinsufficient for SRSF2 and in CD34(+) CMML patient cells harboring SRSF2 mutation. Together, our studies establish an intimate link across telomere biology, aberrant RNA splicing, and myeloid progenitor differentiation.
Asunto(s)
Diferenciación Celular/genética , Hematopoyesis/genética , Síndromes Mielodisplásicos/genética , Telómero , Animales , Haploinsuficiencia , Humanos , Ratones , Síndromes Mielodisplásicos/patología , Proteínas Nucleares/genética , Empalme del ARN , Ribonucleoproteínas/genética , Factores de Empalme Serina-ArgininaRESUMEN
Mutated KRAS (KRAS*) is a fundamental driver in the majority of pancreatic ductal adenocarcinomas (PDAC). Using an inducible mouse model of KRAS*-driven PDAC, we compared KRAS* genetic extinction with pharmacologic inhibition of MEK1 in tumor spheres and in vivo. KRAS* ablation blocked proliferation and induced apoptosis, whereas MEK1 inhibition exerted cytostatic effects. Proteomic analysis evidenced that MEK1 inhibition was accompanied by a sustained activation of the PI3K-AKT-MTOR pathway and by the activation of AXL, PDGFRa, and HER1-2 receptor tyrosine kinases (RTK) expressed in a large proportion of human PDAC samples analyzed. Although single inhibition of each RTK alone or plus MEK1 inhibitors was ineffective, a combination of inhibitors targeting all three coactivated RTKs and MEK1 was needed to inhibit proliferation and induce apoptosis in both mouse and human low-passage PDAC cultures. Importantly, constitutive AKT activation, which may mimic the fraction of AKT2-amplified PDAC, was able to bypass the induction of apoptosis caused by KRAS* ablation, highlighting a potential inherent resistance mechanism that may inform the clinical application of MEK inhibitor therapy. This study suggests that combinatorial-targeted therapies for pancreatic cancer must be informed by the activation state of each putative driver in a given treatment context. In addition, our work may offer explanative and predictive power in understanding why inhibitors of EGFR signaling fail in PDAC treatment and how drug resistance mechanisms may arise in strategies to directly target KRAS.
Asunto(s)
Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Neoplasias Pancreáticas/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Animales , Modelos Animales de Enfermedad , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Neoplasias Pancreáticas/genética , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR/fisiologíaRESUMEN
Nanotechnology involves the engineering of functional systems at nanoscale, thus being attractive for disciplines ranging from materials science to biomedicine. One of the most active research areas of the nanotechnology is nanomedicine, which applies nanotechnology to highly specific medical interventions for prevention, diagnosis, and treatment of diseases, including cancer disease. Over the past two decades, the rapid developments in nanotechnology have allowed the incorporation of multiple therapeutic, sensing, and targeting agents into nanoparticles, for detection, prevention, and treatment of cancer diseases. Nanoparticles offer many advantages as drug carrier systems since they can improve the solubility of poorly water-soluble drugs, modify pharmacokinetics, increase drug half-life by reducing immunogenicity, improve bioavailability, and diminish drug metabolism. They can also enable a tunable release of therapeutic compounds and the simultaneous delivery of two or more drugs for combination therapy. In this review, we discuss the recent advances in the use of different types of nanoparticles for systemic and topical drug delivery in the treatment of skin cancer. In particular, the progress in the treatment with nanocarriers of basal cell carcinoma, squamous cell carcinoma, and melanoma has been reported.