RESUMEN
Epithelial to mesenchymal transition (EMT) in cancer cells has been associated with metastasis, stemness, and resistance to therapy. Some tumors undergo EMT while others do not, which may reflect intrinsic properties of their cell of origin. However, this possibility is largely unexplored. By targeting the same oncogenic mutations to discrete skin compartments, we show that cell-type-specific chromatin and transcriptional states differentially prime tumors to EMT. Squamous cell carcinomas (SCCs) derived from interfollicular epidermis (IFE) are generally well differentiated, while hair follicle (HF) stem cell-derived SCCs frequently exhibit EMT, efficiently form secondary tumors, and possess increased metastatic potential. Transcriptional and epigenomic profiling revealed that IFE and HF tumor-initiating cells possess distinct chromatin landscapes and gene regulatory networks associated with tumorigenesis and EMT that correlate with accessibility of key epithelial and EMT transcription factor binding sites. These findings highlight the importance of chromatin states and transcriptional priming in dictating tumor phenotypes and EMT.
Asunto(s)
Carcinoma de Células Escamosas/patología , Cromatina/metabolismo , Transición Epitelial-Mesenquimal , Células Madre Neoplásicas/patología , Neoplasias Cutáneas/patología , Animales , Secuencia de Bases , Carcinogénesis/patología , Carcinoma de Células Escamosas/genética , Diferenciación Celular , Linaje de la Célula/genética , Células Clonales , Epigénesis Genética , Células Epiteliales/metabolismo , Células Epiteliales/patología , Células HEK293 , Folículo Piloso/patología , Humanos , Ratones , Metástasis de la Neoplasia , Fosfoproteínas/metabolismo , Proteínas Represoras/metabolismo , Neoplasias Cutáneas/genética , Transactivadores/metabolismo , Transcripción GenéticaRESUMEN
Melanoma is a paradigm of aggressive tumors with a complex and heterogeneous genetic background. Still, melanoma cells frequently retain developmental traits that trace back to lineage specification programs. In particular, lysosome-associated vesicular trafficking is emerging as a melanoma-enriched lineage dependency. However, the contribution of other lysosomal functions such as autophagy to melanoma progression is unclear, particularly in the context of metastasis and resistance to targeted therapy. Here we mined a broad spectrum of cancers for a meta-analysis of mRNA expression, copy number variation and prognostic value of 13 core autophagy genes. This strategy identified heterozygous loss of ATG5 at chromosome band 6q21 as a distinctive feature of advanced melanomas. Importantly, partial ATG5 loss predicted poor overall patient survival in a manner not shared by other autophagy factors and not recapitulated in other tumor types. This prognostic relevance of ATG5 copy number was not evident for other 6q21 neighboring genes. Melanocyte-specific mouse models confirmed that heterozygous (but not homozygous) deletion of Atg5 enhanced melanoma metastasis and compromised the response to targeted therapy (exemplified by dabrafenib, a BRAF inhibitor in clinical use). Collectively, our results support ATG5 as a therapeutically relevant dose-dependent rheostat of melanoma progression. Moreover, these data have important translational implications in drug design, as partial blockade of autophagy genes may worsen (instead of counteracting) the malignant behavior of metastatic melanomas.
Asunto(s)
Proteína 5 Relacionada con la Autofagia/genética , Pérdida de Heterocigocidad/genética , Melanoma/genética , Melanoma/patología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Animales , Autofagia/efectos de los fármacos , Autofagia/genética , Mapeo Cromosómico , Variaciones en el Número de Copia de ADN/genética , Metilación de ADN/genética , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Heterocigoto , Ratones , Metástasis de la Neoplasia , Nevo/genética , Nevo/patología , Pigmentación/genética , Pronóstico , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas B-raf/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Riesgo , Análisis de SupervivenciaRESUMEN
True to their inherent aggressive behavior, melanomas keep impressing the melanoma community with their ability to bypass tumor suppressor mechanisms. Name a pathway with the potential to control cell survival and melanoma cells will likely have it potentiated by multiple genetic or epigenetic alterations. In the context of progression and chemoresistance, large efforts have been dedicated to the identification of protective mechanisms associated with or linked to apoptotic death programs. These studies have guided the design of targeted anticancer strategies. Still, the promise for pro-apoptotic inducers as lead compounds for drug development has yet to come to fruition. It was then a question of time to identify alternative modulators of cell viability. An ideal candidate that is raising great expectations in the oncology field is autophagy, a catabolic process with multiple roles in cell homeostasis. Here we review the incipient literature on autophagy markers in melanocytic lesions. Intriguingly, histopathological studies are unveiling an intrinsic inter- and intratumor variability in the expression of autophagy modulators. Nonetheless, functional studies support a key role of autopaphagy programs in the response to a variety of stress factors. These include adaptive responses to nutrient deprivation, hypoxia and many anticancer agents, among other stimuli. Strategies are being also developed to mobilize the endocytic machinery and shift autolysosomes into death effectors. The opportunities that lie ahead in this field are exciting. Various authophagy mediators are potentially druggable. Moreover, animal models and the development of sophisticated screening methods offer a platform for multilevel academic-industrial collaborations. These efforts are expected to open avenues of research and, hopefully, lead to a more rational approach to melanoma treatment.
Asunto(s)
Autofagia , Melanoma/patología , Neoplasias Cutáneas/patología , Animales , Humanos , Melaninas/biosíntesis , Melanoma/inmunología , Melanoma/terapia , Fagosomas/metabolismo , Transducción de Señal , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/terapiaRESUMEN
Inappropriate drug delivery, secondary toxicities, and persistent chemo- and immunoresistance have traditionally compromised treatment response in melanoma. Using cellular systems and genetically engineered mouse models, we show that melanoma cells retain an innate ability to recognize cytosolic double-stranded RNA (dsRNA) and mount persistent stress response programs able to block tumor growth, even in highly immunosuppressed backgrounds. The dsRNA mimic polyinosine-polycytidylic acid, coadministered with polyethyleneimine as carrier, was identified as an unanticipated inducer of autophagy downstream of an exacerbated endosomal maturation program. A concurrent activity of the dsRNA helicase MDA-5 driving the proapoptotic protein NOXA resulted in an efficient autodigestion of melanoma cells. These results reveal tractable links for therapeutic intervention among dsRNA helicases, endo/lysosomes, and apoptotic factors.
Asunto(s)
Apoptosis/inmunología , Autofagia/inmunología , Inmunidad Innata , Melanoma/inmunología , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Autofagia/efectos de los fármacos , Autofagia/genética , Proteína 5 Relacionada con la Autofagia , Línea Celular Tumoral , Células Cultivadas , ARN Helicasas DEAD-box/metabolismo , Endosomas/efectos de los fármacos , Endosomas/genética , Endosomas/metabolismo , Humanos , Helicasa Inducida por Interferón IFIH1 , Lisosomas/efectos de los fármacos , Lisosomas/genética , Lisosomas/metabolismo , Melanoma/patología , Melanoma/terapia , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/fisiología , Fagosomas/efectos de los fármacos , Fagosomas/genética , Fagosomas/metabolismo , Poli C/farmacología , Polietileneimina/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN BicatenarioRESUMEN
Apoptosis is a process of cellular suicide executed by caspases. Impaired activation of caspase-9 may contribute to chemoresistance in cancer. Activation of caspase-9 occurs after binding to Apaf-1 and formation of the apoptosome in the presence of cytochrome c/(d)ATP. We used a proteomics approach to identify proteins in caspase-9-protein complexes in extracts derived from NSCLC cells with(out) cytochrome c/dATP. Using co-immunoprecipitation, one-dimensional gel electrophoresis and tandem mass spectrometry, 38 proteins were identified of which 24 differential interactors. The differential interactors can be functionally assigned to cytoskeletal (re)organization and cell motility, catalytic activity, and transcriptional processes and apoptosis. The interaction of caspase-9 with Apaf-1 was confirmed and acetylserotonin-O-methyltransferase-like protein was identified as a candidate substrate of caspase-9. Novel interactors were found including galectin-3, swiprosin-1 and the membrane-cytoskeleton linkers Ezrin/Radixin/Moesin. Co-immunoprecipitation and Western blot experiments confirmed the interaction of caspase-9 with several identified binding partners. A large number of cytoskeletal proteins associated with unprocessed caspase-9 may indicate a scaffold function of this structure and/or may act as caspase substrates during apoptosis. Together, our results indicate that proteomic analysis of the caspase-9-associated protein complexes is a powerful exploratory approach to identify novel caspase substrates and/or regulators of caspase-9-dependent apoptosis.
Asunto(s)
Apoptosis/fisiología , Caspasa 9/metabolismo , Proteoma/metabolismo , Secuencia de Aminoácidos , Línea Celular Tumoral , Citocromos c/metabolismo , Nucleótidos de Desoxiadenina/farmacología , Humanos , Datos de Secuencia Molecular , Péptidos/análisis , Unión Proteica , Espectrometría de Masas en Tándem/métodosRESUMEN
Bortezomib is a novel anti-cancer agent which has shown promising activity in non-small lung cancer (NSCLC) patients. However, only a subset of patients respond to this treatment. We show that NSCLC cell lines are differentially sensitive to bortezomib, IC50 values ranging from 5 to 83 nM. The apoptosis-inducing potential of bortezomib in NSCLC cells was found to be dependent not only on the apoptotic phenotype but also on the proteasomal phenotype of individual cell lines. Upon effective proteasome inhibition, H460 cells were more susceptible to apoptosis induction by bortezomib than SW1573 cells, indicating a different apoptotic phenotype. However, exposure to a low dose of bortezomib did only result in SW1573 cells, and not in H460 cells, in inhibition of proteasome activity and subsequent apoptosis. This suggests a different proteasomal phenotype as well. Additionally, overexpression of anti-apoptotic protein Bcl-2 in H460 cells did not affect the proteasomal phenotype of H460 cells but did result in decreased bortezomib-induced apoptosis. In conclusion, successful proteasome-inhibitor based treatment strategies in NSCLC face the challenge of having to overcome apoptosis resistance as well as proteasomal resistance of individual lung cancer cells. Further studies in NSCLC are warranted to elucidate underlying mechanisms.
Asunto(s)
Apoptosis/efectos de los fármacos , Ácidos Borónicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/patología , Complejo de la Endopetidasa Proteasomal/metabolismo , Pirazinas/farmacología , Bortezomib , Dominio Catalítico , Línea Celular Tumoral , Humanos , Sensibilidad y EspecificidadRESUMEN
Defects in the apoptotic machinery may contribute to chemoresistance of non-small cell lung cancer (NSCLC) cells. We have previously showed a deficiency in mitochondria-dependent caspase-9 activation in NSCLC H460 cells after exposure to cisplatin, a drug widely used to treat NSCLC. Here we show that, unlike cisplatin, the novel anticancer agent bortezomib efficiently induces caspase-9 activation and apoptosis in H460 cells. A comparative analysis of molecular events underlying cell death in bortezomib-treated versus cisplatin-treated H460 cells revealed that bortezomib, but not cisplatin, caused a rapid and abundant release of cytochrome c and Smac/DIABLO from mitochondria. This was associated with a marked increase in levels of the BH3-only proapoptotic protein Noxa and the antiapoptotic protein Mcl-1. Taken together, our data show that bortezomib, by promoting a proapoptotic shift in the levels of proteins involved in mitochondrial outer-membrane permeabilization, is a potent activator of the mitochondrial pathway of apoptosis in NSCLC cells. Our preclinical results support further investigation of bortezomib-based therapies as a possible new treatment modality for NSCLC.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Ácidos Borónicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/patología , Cisplatino/farmacología , Neoplasias Pulmonares/patología , Mitocondrias/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Pirazinas/farmacología , Western Blotting , Bortezomib , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Inhibidores de Caspasas , Caspasas/metabolismo , Citocromos c/metabolismo , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Mitocondrias/metabolismo , Células Tumorales Cultivadas/efectos de los fármacos , Regulación hacia ArribaRESUMEN
X-linked IAP (XIAP) suppresses apoptosis by binding to initiator caspase-9 and effector caspases-3 and -7. Smac/DIABLO that is released from mitochondria during apoptosis can relieve its inhibitory activity. Here we investigated the role of XIAP in the previously found obstruction of chemotherapy-induced caspase-9 activation in non-small cell lung cancer (NSCLC) cells. Endogenously expressed XIAP bound active forms of both caspase-9 and caspase-3. However, downregulation of XIAP using shRNA or disruption of XIAP/caspase-9 interaction using a small molecule Smac mimic were unable to significantly induce caspase-9 activity, indicating that despite a strong binding potential of XIAP to caspase-9 it is not a major determinant in blocking caspase-9 in NSCLC cells. Although unable to revert caspase-9 blockage, the Smac mimic was able to enhance cisplatin-induced apoptosis, which was accompanied by increased caspase-3 activity. Additionally, a more detailed analysis of caspase activation in response to cisplatin indicated a reverse order of activation, whereby caspase-3 cleaved caspase-9 yielding an inactive form. Our findings indicate that the use of small molecule Smac mimic, when combined with an apoptotic trigger, may have therapeutic potential for the treatment of NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Caspasa 3/metabolismo , Cisplatino/farmacología , Regulación Neoplásica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/fisiología , Neoplasias Pulmonares/metabolismo , Proteínas Mitocondriales/fisiología , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Antineoplásicos/farmacología , Apoptosis , Proteínas Reguladoras de la Apoptosis , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Caspasa 9/metabolismo , Caspasa 9/fisiología , Línea Celular Tumoral , Regulación hacia Abajo , Activación Enzimática , Regulación Enzimológica de la Expresión Génica , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Modelos Biológicos , Interferencia de ARN , Transducción de Señal , Proteína Inhibidora de la Apoptosis Ligada a X/fisiologíaRESUMEN
BACKGROUND: TUCAN is a caspase recruitment domain (CARD)-containing protein involved in tumor biology by regulating apoptosis and the NFkappaB pathway. Inhibition of caspase-9 may cause drug resistance. The pattern of expression, localization and prognostic value of TUCAN in the tumors of patients with non-small cell lung cancer (NSCLC) treated with chemotherapy were assessed in this study. MATERIALS AND METHODS: Using immunohistochemistry, the expression and localization of TUCAN was evaluated in forty-nine tumor specimens from patients with NSCLC who underwent neoadjuvant chemotherapy (32 stage IIB or IIIA), or palliative chemotherapy (17 stage IIIB or IV). The correlation between TUCAN expression and subcellular localization, major patient characteristics, response to the treatment and overall survival were assessed. RESULTS: TUCAN expression was detectable in 34 out of 49 (69%) tumor specimens. Among the positively-stained specimens, three patterns of localization were observed: 5 samples (11%) showed exclusive nuclear localization, 13 samples (27%) contained only cytoplasmic staining and 15 (31%) showed both cytoplasmic and nuclear localization. There was no significant correlation between the localization of TUCAN and response to chemotherapy. Although TUCAN expression was not correlated with outcome, interestingly, exclusive cytoplasmic localization of TUCAN predicted shorter survival (p = 0.027). CONCLUSION: Our results suggest that differential localization of TUCAN may be a prognostic factor for NSCLC, despite the lack of predictive value for response to chemotherapy.
Asunto(s)
Antineoplásicos/uso terapéutico , Proteínas Adaptadoras de Señalización CARD/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/metabolismo , Adulto , Anciano , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Pronóstico , Fracciones Subcelulares/metabolismoRESUMEN
BACKGROUND: Activation of caspase-9 in response to treatment with cytotoxic drugs is inhibited in NSCLC cells, which may contribute to the clinical resistance to chemotherapy shown in this type of tumor. The aim of the present study was to investigate the mechanism of caspase-9 inhibition, with a focus on a possible role of TUCAN as caspase-9 inhibitor and a determinant of chemosensitivity in NSCLC cells. METHODS: Caspase-9 processing and activation were investigated by Western blot and by measuring the cleavage of the fluorogenic substrate LEHD-AFC. Proteins interaction assays, and RNA interference in combination with cell viability and apoptosis assays were used to investigate the involvement of TUCAN in inhibition of caspase-9 and chemosensitivity NSCLC. RESULTS: Analysis of the components of the caspase-9 activation pathway in a panel of NSCLC and SCLC cells revealed no intrinsic defects. In fact, exogenously added cytochrome c and dATP triggered procaspase-9 cleavage and activation in lung cancer cell lysates, suggesting the presence of an inhibitor. The reported inhibitor of caspase-9, TUCAN, was exclusively expressed in NSCLC cells. However, interactions between TUCAN and procaspase-9 could not be demonstrated by any of the assays used. Furthermore, RNA interference-mediated down-regulation of TUCAN did not restore cisplatin-induced caspase-9 activation or affect cisplatin sensitivity in NSCLC cells. CONCLUSION: These results indicate that procaspase-9 is functional and can undergo activation and full processing in lung cancer cell extracts in the presence of additional cytochrome c/dATP. However, the inhibitory protein TUCAN does not play a role in inhibition of procaspase-9 and in determining the sensitivity to cisplatin in NSCLC.