RESUMEN
In breast cancers, aberrant activation of the RAS/MAPK pathway is strongly associated with mesenchymal features and stemness traits, suggesting an interplay between this mitogenic signaling pathway and epithelial-to-mesenchymal plasticity (EMP). By using inducible models of human mammary epithelial cells, we demonstrate herein that the oncogenic activation of RAS promotes ZEB1-dependent EMP, which is necessary for malignant transformation. Notably, EMP is triggered by the secretion of pro-inflammatory cytokines from neighboring RAS-activated senescent cells, with a prominent role for IL-6 and IL-1α. Our data contrast with the common view of cellular senescence as a tumor-suppressive mechanism and EMP as a process promoting late stages of tumor progression in response to signals from the tumor microenvironment. We highlighted here a pro-tumorigenic cooperation of RAS-activated mammary epithelial cells, which leverages on oncogene-induced senescence and EMP to trigger cellular reprogramming and malignant transformation.
Asunto(s)
Carcinogénesis , Transformación Celular Neoplásica , Humanos , Carcinogénesis/genética , Transformación Celular Neoplásica/genética , Mama , Genes ras , Transducción de Señal , Senescencia Celular/genética , Microambiente TumoralRESUMEN
INTRODUCTION: Epithelial-to-mesenchymal transition (EMT) is associated with tumor aggressiveness, drug resistance, and poor survival in non-small cell lung cancer (NSCLC) and other cancers. The identification of immune-checkpoint ligands (ICPLs) associated with NSCLCs that display a mesenchymal phenotype (mNSCLC) could help to define subgroups of patients who may benefit from treatment strategies using immunotherapy. METHODS: We evaluated ICPL expression in silico in 130 NSCLC cell lines. In vitro, CRISPR/Cas9-mediated knockdown and lentiviral expression were used to assess the impact of ZEB1 expression on CD70. Gene expression profiles of lung cancer samples from the TCGA (n = 1018) and a dataset from MD Anderson Cancer Center (n = 275) were analyzed. Independent validation was performed by immunohistochemistry and targeted-RNA sequencing in 154 NSCLC whole sections, including a large cohort of pulmonary sarcomatoid carcinomas (SC, n = 55). RESULTS: We uncover that the expression of CD70, a regulatory ligand from the tumor necrosis factor ligand family, is enriched in mNSCLC in vitro models. Mechanistically, the EMT-inducer ZEB1 impacted CD70 expression and fostered increased activity of the CD70 promoter. CD70 overexpression was also evidenced in mNSCLC patient tumor samples and was particularly enriched in SC, a lung cancer subtype associated with poor prognosis. In these tumors, CD70 expression was associated with decreased CD3+ and CD8+ T-cell infiltration and increased T-cell exhaustion markers. CONCLUSION: Our results provide evidence on the pivotal roles of CD70 and ZEB1 in immune escape in mNSCLC, suggesting that EMT might promote cancer progression and metastasis by not only increasing cancer cell plasticity but also reprogramming the immune response in the local tumor microenvironment.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Ligando CD27/genética , Ligando CD27/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Ligandos , Neoplasias Pulmonares/patología , Microambiente TumoralRESUMEN
A characteristic of cancer development is the acquisition of genomic instability, which results from the inaccurate repair of DNA damage. Among double-strand break repair mechanisms induced by oncogenic stress, the highly mutagenic theta-mediated end-joining (TMEJ) pathway, which requires DNA polymerase theta (POLθ) encoded by the POLQ gene, has been shown to be overexpressed in several human cancers. However, little is known regarding the regulatory mechanisms of TMEJ and the consequence of its dysregulation. In this study, we combined a bioinformatics approach exploring both Molecular Taxonomy of Breast Cancer International Consortium and The Cancer Genome Atlas databases with CRISPR/Cas9-mediated depletion of the zinc finger E-box binding homeobox 1 (ZEB1) in claudin-low tumor cells or forced expression of ZEB1 in basal-like tumor cells, two triple-negative breast cancer (TNBC) subtypes, to demonstrate that ZEB1 represses POLQ expression. ZEB1, a master epithelial-to-mesenchymal transition-inducing transcription factor, interacted directly with the POLQ promoter. Moreover, downregulation of POLQ by ZEB1 fostered micronuclei formation in TNBC tumor cell lines. Consequently, ZEB1 expression prevented TMEJ activity, with a major impact on genome integrity. In conclusion, we showed that ZEB1 directly inhibits the expression of POLQ and, therefore, TMEJ activity, controlling both stability and integrity of breast cancer cell genomes. SIGNIFICANCE: These findings uncover an original mechanism of TMEJ regulation, highlighting ZEB1 as a key player in genome stability during cancer progression via its repression of POLQ.See related commentary by Carvajal-Maldonado and Wood, p. 1441.
Asunto(s)
Neoplasias de la Mama , Factores de Transcripción , Neoplasias de la Mama/genética , Línea Celular Tumoral , Transición Epitelial-Mesenquimal/genética , Femenino , Humanos , Mutágenos , Factores de Transcripción/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genéticaRESUMEN
The study aims to combine the delivery of two anticancer drugs to target both proliferating cancer cells and dormant cancer stem cells (CSCs) present in colorectal cancer. Two drugs were selected and encapsulated in lipid nanocapsules: SN38, the active form of irinotecan, which is unstable in the plasma but active against replicating cells, and salinomycin, a highly toxic ionophore active against cancer stem cells that is not suitable for clinical use. Using an engineered medium that enhanced the ratio of CSCs in HCT116 cell cultures, we demonstrated by clonogenicity tests and in sphere assays that Salinomycin acts mainly on CSCs, while SN38 acts mainly on proliferating cancer cells. In a preclinical murine CRC model, encapsulation of both drugs in lipid nanocapsules reduced their toxicity, including hemolysis, and led to a higher survival than what was observed following treatment with single drugs or non-encapsulated drugs. Nanoparticles loaded with an anticancer drug and salinomycin were effective against the therapy-resistant dormant CSCs and cancer cells.
Asunto(s)
Neoplasias Colorrectales , Nanopartículas , Animales , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Irinotecán , Ratones , Nanomedicina , Células Madre Neoplásicas , PiranosRESUMEN
Despite advances in single-cell and molecular techniques, it is still unclear how to best quantify phenotypic heterogeneity in cancer cells that evolved beyond normal, known classifications. We present an approach to phenotypically characterize cells based on their activities rather than static classifications. We validated the detectability of specific activities (epithelial-mesenchymal transition, glycolysis) in single cells, using targeted RT-qPCR analyses and in vitro inductions. We analyzed 50 established activity signatures as a basis for phenotypic description in public data and computed cell-cell distances in 28,513 cells from 85 patients and 8 public datasets. Despite not relying on any classification, our measure correlated with standard diversity indices in populations of known structure. We identified bottlenecks as phenotypic diversity reduced upon colorectal cancer initiation. This suggests that focusing on what cancer cells do rather than what they are can quantify phenotypic diversity in universal fashion, to better understand and predict intra-tumor heterogeneity dynamics.
RESUMEN
Chromosomal instability (CIN), a feature of most adult neoplasms from their early stages onward, is a driver of tumorigenesis. However, several malignancy subtypes, including some triple-negative breast cancers, display a paucity of genomic aberrations, thus suggesting that tumor development may occur in the absence of CIN. Here we show that the differentiation status of normal human mammary epithelial cells dictates cell behavior after an oncogenic event and predetermines the genetic routes toward malignancy. Whereas oncogene induction in differentiated cells induces massive DNA damage, mammary stem cells are resistant, owing to a preemptive program driven by the transcription factor ZEB1 and the methionine sulfoxide reductase MSRB3. The prevention of oncogene-induced DNA damage precludes induction of the oncosuppressive p53-dependent DNA-damage response, thereby increasing stem cells' intrinsic susceptibility to malignant transformation. In accord with this model, a subclass of breast neoplasms exhibit unique pathological features, including high ZEB1 expression, a low frequency of TP53 mutations and low CIN.
Asunto(s)
Neoplasias de la Mama/genética , Carcinoma/genética , Diferenciación Celular/genética , Células Epiteliales/metabolismo , Regulación Neoplásica de la Expresión Génica , Inestabilidad Genómica/genética , Metionina Sulfóxido Reductasas/genética , Células Madre/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Neoplasias de la Mama/metabolismo , Carcinoma/metabolismo , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Inmunoprecipitación de Cromatina , Daño del ADN , Células Epiteliales/citología , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Humanos , Immunoblotting , Glándulas Mamarias Humanas/citología , Metionina Sulfóxido Reductasas/metabolismo , Ratones Endogámicos NOD , Persona de Mediana Edad , Especies Reactivas de Oxígeno , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADN , Células Madre/citología , Análisis de Matrices Tisulares , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Adulto Joven , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismoRESUMEN
By fostering cell commitment to the epithelial-to-mesenchymal transition (EMT), SNAIL proteins endow cells with motility, thereby favoring the metastatic spread of tumor cells. Whether the phenotypic change additionally facilitates tumor initiation has never been addressed. Here we demonstrate that when a SNAIL protein is ectopically produced in non-transformed mammary epithelial cells, the cells are protected from anoikis and proliferate under low-adherence conditions: a hallmark of cancer cells. The three SNAIL proteins show unequal oncogenic potential, strictly correlating with their ability to promote EMT. SNAIL3 especially behaves as a poor EMT-inducer comforting the concept that the transcription factor functionally diverges from its two related proteins.
Asunto(s)
Transformación Celular Neoplásica/patología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Transición Epitelial-Mesenquimal , Glándulas Mamarias Humanas/patología , Factores de Transcripción/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Adhesión Celular/genética , Supervivencia Celular/genética , Transformación Celular Neoplásica/genética , Transición Epitelial-Mesenquimal/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción de la Familia Snail , Telomerasa/metabolismo , Factores de Transcripción/genéticaRESUMEN
As solid tumors expand, oxygen and nutrients become limiting owing to inadequate vascularization and diffusion. How malignant cells cope with this potentially lethal metabolic stress remains poorly understood. We found that glucose shortage associated with malignant progression triggers apoptosis through the endoplasmic reticulum (ER) unfolded protein response (UPR). ER stress is in part caused by reduced glucose flux through the hexosamine pathway. Deletion of the proapoptotic UPR effector CHOP in a mouse model of K-ras(G12V)-induced lung cancer increases tumor incidence, strongly supporting the notion that ER stress serves as a barrier to malignancy. Overcoming this barrier requires the selective attenuation of the PERK-CHOP arm of the UPR by the molecular chaperone p58(IPK). Furthermore, p58(IPK)-mediated adaptive response enables cells to benefit from the protective features of chronic UPR. Altogether, these results show that ER stress activation and p58(IPK) expression control the fate of malignant cells facing glucose shortage.
Asunto(s)
Apoptosis , Transformación Celular Neoplásica/metabolismo , Glucosa/deficiencia , Chaperonas Moleculares/fisiología , Factor de Transcripción CHOP/metabolismo , eIF-2 Quinasa/metabolismo , Acetilgalactosamina/metabolismo , Animales , Hipoxia de la Célula , Línea Celular , Proliferación Celular , Factor Neurotrófico Derivado de la Línea Celular Glial/fisiología , Transportador de Glucosa de Tipo 1/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Ácido Láctico/metabolismo , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Proteínas Proto-Oncogénicas c-ret/metabolismo , Ratas , Respuesta de Proteína DesplegadaRESUMEN
The epithelial-mesenchymal transition (EMT) is an embryonic transdifferentiation process consisting of conversion of polarized epithelial cells to motile mesenchymal ones. EMT-inducing transcription factors are aberrantly expressed in multiple tumor types and are known to favor the metastatic dissemination process. Supporting oncogenic activity within primary lesions, the TWIST and ZEB proteins can prevent cells from undergoing oncogene-induced senescence and apoptosis by abolishing both p53- and RB-dependent pathways. Here we show that they also downregulate PP2A phosphatase activity and efficiently cooperate with an oncogenic version of H-RAS in malignant transformation of human mammary epithelial cells. Thus, by down-regulating crucial tumor suppressor functions, EMT inducers make cells particularly prone to malignant conversion. Importantly, by analyzing transformed cells generated in vitro and by characterizing novel transgenic mouse models, we further demonstrate that cooperation between an EMT inducer and an active form of RAS is sufficient to trigger transformation of mammary epithelial cells into malignant cells exhibiting all the characteristic features of claudin-low tumors, including low expression of tight and adherens junction genes, EMT traits, and stem cell-like characteristics. Claudin-low tumors are believed to be the most primitive breast malignancies, having arisen through transformation of an early epithelial precursor with inherent stemness properties and metaplastic features. Challenging this prevailing view, we propose that these aggressive tumors arise from cells committed to luminal differentiation, through a process driven by EMT inducers and combining malignant transformation and transdifferentiation.
Asunto(s)
Neoplasias de la Mama , Transformación Celular Neoplásica , Claudinas , Transición Epitelial-Mesenquimal , Glándulas Mamarias Humanas/metabolismo , Proteína Fosfatasa 2 , Proteína 1 Relacionada con Twist/genética , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Diferenciación Celular , Línea Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Claudinas/genética , Claudinas/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Transición Epitelial-Mesenquimal/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Genes ras , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Humanas/citología , Ratones , Ratones Transgénicos , Proteína Fosfatasa 2/antagonistas & inhibidores , Proteína Fosfatasa 2/metabolismo , Proteína de Retinoblastoma/metabolismo , Telomerasa/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteína 1 Relacionada con Twist/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
Despite its frequent inactivation in human breast cancers, the role of p21(Cip1) (p21) in morphological plasticity of normal mammary epithelial cells is still poorly understood. To address this question, we have investigated the consequences of p21 silencing in two-dimensional (2D) morphogenesis of untransformed human mammary epithelial cells. Here we show that p21 inactivation causes a reduction of 2D cell spreading and suppresses focal adhesion. In order to investigate the cytoskeletal modifications associated with this altered morphology, we have analyzed the microtubule dynamics in interphase p21-depleted cells. Our results demonstrate that interphase microtubule dynamic instability is strongly increased by p21 silencing. This alteration correlates with severe microtubule hypoacetylation. Next, we show that these microtubule defects in p21-depleted cells can be reversed by the use of the small molecule tubacin, a specific inhibitor of the α-tubulin deacetylase HDAC6. Tubacin-induced microtubule dynamics decrease also correlates with a partial recovery of cell spreading and focal adhesion in those cells. Collectively, these data indicate that p21 regulates the morphological plasticity of normal mammary epithelial cells by modulating dynamics of key cytoskeletal components.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Células Epiteliales/metabolismo , Adhesiones Focales/metabolismo , Interfase , Glándulas Mamarias Humanas/metabolismo , Microtúbulos/metabolismo , Anilidas/farmacología , Adhesión Celular , Proliferación Celular , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Citoesqueleto/metabolismo , ADN/biosíntesis , Células Epiteliales/citología , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Histona Desacetilasa 6 , Histona Desacetilasas/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Glándulas Mamarias Humanas/citología , Proteínas de Unión al GTP rho/antagonistas & inhibidoresRESUMEN
Twist1 and Twist2 are major regulators of embryogenesis. Twist1 has been shown to favor the metastatic dissemination of cancer cells through its ability to induce an epithelial-mesenchymal transition (EMT). Here, we show that a large fraction of human cancers overexpress Twist1 and/or Twist2. Both proteins override oncogene-induced premature senescence by abrogating key regulators of the p53- and Rb-dependent pathways. Twist1 and Twist2 cooperate with Ras to transform mouse embryonic fibroblasts. Interestingly, in epithelial cells, the oncogenic cooperation between Twist proteins and activated mitogenic oncoproteins, such as Ras or ErbB2, leads to complete EMT. These findings suggest an unanticipated direct link between early escape from failsafe programs and the acquisition of invasive features by cancer cells.