RESUMEN

One of the hurdles to the development of new anticancer therapies is the lack of in vitro models which faithfully reproduce the in vivo tumor microenvironment (TME). Understanding the dynamic relationships between the components of the TME in a controllable, scalable, and reliable setting would indeed support the discovery of biological targets impacting cancer diagnosis and therapy. Cancer research is increasingly shifting from traditional two-dimensional (2D) cell culture toward three-dimensional (3D) culture models, which have been demonstrated to increase the significance and predictive value of in vitro data. In this scenario, microphysiological systems (also known as organs-on-chip) have emerged as a relevant technological platform enabling more predictive investigation of cell-cell and cell-ECM interplay in cancer, attracting a significant research effort in the last years. This review illustrates one decade of progress in the field of tumor-microenvironment-on-chip (TMOC) approaches, exploiting either cell-laden microfluidic chambers or microfluidic confined tumor spheroids to model the TME. TMOCs have been designed to recapitulate several aspects of the TME, including tumor cells, the tumor-associated stroma, the immune system, and the vascular component. Significantly, the last aspect has emerged for its pivotal role in orchestrating cellular interactions and modulating drug pharmacokinetics on-chip. A further advancement has been represented by integration of TMOCs into multi-organ microphysiological systems, with the final aim to follow the metastatic cascade to target organs and to study the effects of chemotherapies at a systemic level. We highlight that the increased degree of complexity achieved by the most advanced TMOC models has enabled scientists to shed new light on the role of microenvironmental factors in tumor progression, metastatic cascade, and response to drugs.

Asunto(s)

Neoplasias , Humanos , Neoplasias/patología , Microfluídica , Microambiente Tumoral , Técnicas de Cultivo de Célula

RESUMEN

This corrects the article DOI: 10.1038/onc.2017.75.

RESUMEN

Recent evidences suggest that stearoyl-CoA-desaturase 1 (SCD1), the enzyme involved in monounsaturated fatty acids synthesis, has a role in several cancers. We previously demonstrated that SCD1 is important in lung cancer stem cells survival and propagation. In this article, we first show, using primary cell cultures from human lung adenocarcinoma, that the effectors of the Hippo pathway, Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), are required for the generation of lung cancer three-dimensional cultures and that SCD1 knock down and pharmacological inhibition both decrease expression, nuclear localization and transcriptional activity of YAP and TAZ. Regulation of YAP/TAZ by SCD1 is at least in part dependent upon ß-catenin pathway activity, as YAP/TAZ downregulation induced by SCD1 blockade can be rescued by the addition of exogenous wnt3a ligand. In addition, SCD1 activation of nuclear YAP/TAZ requires inactivation of the ß-catenin destruction complex. In line with the in vitro findings, immunohistochemistry analysis of lung adenocarcinoma samples showed that expression levels of SCD1 co-vary with those of ß-catenin and YAP/TAZ. Mining available gene expression data sets allowed to observe that high co-expression levels of SCD1, ß-catenin, YAP/TAZ and downstream targets have a strong negative prognostic value in lung adenocarcinoma. Finally, bioinformatics analyses directed to identify which gene combinations had synergistic effects on clinical outcome in lung cancer showed that poor survival is associated with high co-expression of SCD1, ß-catenin and the YAP/TAZ downstream target birc5. In summary, our data demonstrate for the first time the involvement of SCD1 in the regulation of the Hippo pathway in lung cancer, and point to fatty acids metabolism as a key regulator of lung cancer stem cells.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenocarcinoma/metabolismo , Núcleo Celular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias Pulmonares/metabolismo , Células Madre Neoplásicas/metabolismo , Fosfoproteínas/metabolismo , Estearoil-CoA Desaturasa/metabolismo , Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Complejo de Señalización de la Axina/metabolismo , Regulación hacia Abajo , Ácidos Grasos/metabolismo , Femenino , Células HEK293 , Vía de Señalización Hippo , Humanos , Inmunohistoquímica , Proteínas Inhibidoras de la Apoptosis/metabolismo , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Masculino , Proteínas de Neoplasias/metabolismo , Cultivo Primario de Células , Pronóstico , Proteínas Serina-Treonina Quinasas/metabolismo , Estabilidad Proteica , ARN Mensajero/metabolismo , Estearoil-CoA Desaturasa/antagonistas & inhibidores , Estearoil-CoA Desaturasa/genética , Survivin , Transactivadores , Factores de Transcripción , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Proteína Wnt3A/metabolismo , Proteínas Señalizadoras YAP

RESUMEN

In recent years, studies of cancer development and recurrence have been influenced by the cancer stem cells (CSCs)/cancer-initiating cells (CICs) hypothesis. According to this, cancer is sustained by highly positioned, chemoresistant cells with extensive capacity of self renewal, which are responsible for disease relapse after chemotherapy. Growth of cancer cells as three-dimensional non-adherent spheroids is regarded as a useful methodology to enrich for cells endowed with CSC-like features. We have recently reported that cell cultures derived from malignant pleural effusions (MPEs) of patients affected by adenocarcinoma of the lung are able to efficiently form spheroids in non-adherent conditions supplemented with growth factors. By expression profiling, we were able to identify a set of genes whose expression is significantly upregulated in lung tumor spheroids versus adherent cultures. One of the most strongly upregulated gene was stearoyl-CoA desaturase (SCD1), the main enzyme responsible for the conversion of saturated into monounsaturated fatty acids. In the present study, we show both by RNA interference and through the use of a small molecule inhibitor that SCD1 is required for lung cancer spheroids propagation both in stable cell lines and in MPE-derived primary tumor cultures. Morphological examination and image analysis of the tumor spheroids formed in the presence of SCD1 inhibitors showed a different pattern of growth characterized by irregular cell aggregates. Electron microscopy revealed that the treated spheroids displayed several features of cellular damage and immunofluorescence analysis on optical serial sections showed apoptotic cells positive for the M30 marker, most of them positive also for the stemness marker ALDH1A1, thus suggesting that the SCD1 inhibitor is selectively killing cells with stem-like properties. Furthermore, SCD1-inhibited lung cancer cells were strongly impaired in their in vivo tumorigenicity and ALDH1A1 expression. These results suggest that SCD1 is a critical target in lung cancer tumor-initiating cells.

Asunto(s)

Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Células Madre Neoplásicas/enzimología , Células Madre Neoplásicas/patología , Estearoil-CoA Desaturasa/metabolismo , Aldehído Deshidrogenasa/genética , Aldehído Deshidrogenasa/metabolismo , Familia de Aldehído Deshidrogenasa 1 , Anoicis/fisiología , Humanos , Derrame Pleural Maligno/metabolismo , Derrame Pleural Maligno/patología , Retinal-Deshidrogenasa , Estearoil-CoA Desaturasa/genética

RESUMEN

It is of great interest for gene therapy to develop vectors that drive the insertion of a therapeutic gene into a chosen specific site on the cellular genome. Adeno-associated virus (AAV) is unique among mammalian viruses in that it integrates into a distinct region of human chromosome 19 (integration site AAVS1). The inverted terminal repeats (ITRs) flanking the AAV genome and the AAV-encoded nonstructural proteins Rep78 and/or Rep68 are the only viral elements necessary and sufficient for site-specific integration. However, it is also known that unrestrained Rep activity may cause nonspecific genomic rearrangements at AAVS1 and/or have detrimental effects on cell physiology. In this paper we describe the generation of a ligand-dependent form of Rep, obtained by fusing a C-terminally deleted Rep68 with a truncated form of the hormone binding domain of the human progesterone receptor, which does not bind progesterone but binds only its synthetic antagonist RU486. The activity of this chimeric protein, named Rep1-491/P, is highly dependent on RU486 in various assays: in particular, it triggers site-specific integration at AAVS1 of an ITR-flanked cassette in a ligand-dependent manner, as efficiently as wild-type Rep68 but without generating unwanted genomic rearrangement at AAVS1.

Asunto(s)

Cromosomas Humanos Par 19 , ADN Helicasas/genética , Proteínas de Unión al ADN , Dependovirus/genética , Transactivadores/genética , Integración Viral , Línea Celular , Núcleo Celular/metabolismo , ADN Helicasas/metabolismo , Replicación del ADN/genética , Vectores Genéticos , Células HeLa , Humanos , Ligandos , Mifepristona/farmacología , Receptores de Progesterona/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transactivadores/metabolismo

RESUMEN

Adeno-associated virus (AAV) integrates very efficiently into a specific site (AAVS1) of human chromosome 19. Two elements of the AAV genome are sufficient: the inverted terminal repeats (ITRs) and the Rep78 or Rep68 protein. The incorporation of the AAV integration machinery in nonviral delivery systems is of great interest for gene therapy. We demonstrate that purified recombinant Rep68 protein is functionally active when directly delivered into human cells by using the polycationic liposome Lipofectamine, promoting the rescue-replication of a codelivered ITR-flanked cassette in adenovirus-infected cells and its site-specific integration in noninfected cells. The sequencing of cloned virus-host DNA junctions confirmed that lipofected Rep68 protein triggers site-specific integration at the same sites in chromosome 19 already characterized in cells latently infected with AAV.

Asunto(s)

Resinas de Intercambio de Catión , Cromosomas Humanos Par 19 , Proteínas de Unión al ADN/metabolismo , Dependovirus/metabolismo , Portadores de Fármacos , Lípidos , Secuencias Repetitivas de Ácidos Nucleicos , Proteínas Virales/metabolismo , Proteínas de Unión al ADN/genética , Células HeLa , Humanos , Células Tumorales Cultivadas , Proteínas Virales/genética , Integración Viral

RESUMEN

We tested the ability of recombinant hMutSalpha (hMSH2/hMSH6) and hMutSbeta (hMSH2/hMSH3) heterodimers to complement the mismatch repair defect of HEC59, a human cancer cell line whose extracts lack all three MutS homologues. Although repair of both base/base mispairs and insertion-deletion loops was restored by hMutSalpha, only the latter substrates were addressed in extracts supplemented with hMutSbeta. hMutSalpha was also able to complement a defect in the repair of base/base mispairs in CHO R and HL60R cell extracts. In these cells, methotrexate-induced amplification of the dihydrofolate reductase (DHFR) locus, which also contains the MSH3 gene, led to an overexpression of MSH3 and thus to a dramatic change in the relative levels of MutSalpha and MutSbeta. As a rule, MSH2 is primarily complexed with MSH6. MutSalpha is thus relatively abundant in mammalian cell extracts, whereas MutSbeta levels are generally low. In contrast, in cells that overexpress MSH3, the available MSH2 protein is sequestered predominantly into MutSbeta. This leads to degradation of the partnerless MSH6 and depletion of MutSalpha. CHO R and HL60R cells therefore lack correction of base/base mispairs, whereas loop repair is maintained by MutSbeta. Consequently, frameshift mutations in CHO R are rare, whereas transitions and transversions are acquired at a rate two orders of magnitude above background. Our data thus support and extend the findings of Drummond et al. [Drummond, J. T., Genschel, J., Wolf, E. & Modrich, P. (1997) Proc. Natl. Acad. Sci. USA 94, 10144-10149] and demonstrate that mismatch repair deficiency can arise not only through mutation or transcriptional silencing of a mismatch repair gene, but also as a result of imbalance in the relative amounts of the MSH3 and MSH6 proteins.

Asunto(s)

Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Ácidos Nucleicos Heterodúplex , Animales , Antimetabolitos Antineoplásicos/farmacología , Células CHO , Cricetinae , Resistencia a Antineoplásicos , Prueba de Complementación Genética , Células HL-60 , Células HeLa , Humanos , Metotrexato/farmacología , Proteína 3 Homóloga de MutS , Proteínas Recombinantes/genética , Tetrahidrofolato Deshidrogenasa/genética