RESUMEN
Intratumoral heterogeneity is a hallmark of glioblastoma multiforme and thought to negatively affect treatment efficacy. Here, we establish libraries of glioma-initiating cell (GIC) clones from patient samples and find extensive molecular and phenotypic variability among clones, including a range of responses to radiation and drugs. This widespread variability was observed as a continuum of multitherapy resistance phenotypes linked to a proneural-mesenchymal shift in the transcriptome. Multitherapy resistance was associated with a semi-stable cell state that was characterized by an altered DNA methylation pattern at promoter regions of mesenchymal master regulators and enhancers. The gradient of cell states within the GIC compartment constitutes a distinct form of heterogeneity. Our findings may open an avenue toward the development of new therapeutic rationales designed to reverse resistant cell states.
Asunto(s)
Metilación de ADN/genética , Glioblastoma/genética , Proteínas de Neoplasias/genética , Células Madre Neoplásicas/metabolismo , Línea Celular Tumoral , Metilación de ADN/efectos de los fármacos , Metilación de ADN/efectos de la radiación , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de la radiación , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Glioblastoma/radioterapia , Humanos , Células Madre Neoplásicas/patología , Regiones Promotoras GenéticasRESUMEN
Glioblastoma (GBM) is the most frequent and malignant form of primary brain tumor. GBM is essentially incurable and its resistance to therapy is attributed to a subpopulation of cells called glioma stem cells (GSCs). To meet the present shortage of relevant GBM cell (GC) lines we developed a library of annotated and validated cell lines derived from surgical samples of GBM patients, maintained under conditions to preserve GSC characteristics. This collection, which we call the Human Glioblastoma Cell Culture (HGCC) resource, consists of a biobank of 48 GC lines and an associated database containing high-resolution molecular data. We demonstrate that the HGCC lines are tumorigenic, harbor genomic lesions characteristic of GBMs, and represent all four transcriptional subtypes. The HGCC panel provides an open resource for in vitro and in vivo modeling of a large part of GBM diversity useful to both basic and translational GBM research.
Asunto(s)
Bancos de Muestras Biológicas , Glioblastoma/patología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Biomarcadores de Tumor , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica , Análisis por Conglomerados , Variaciones en el Número de Copia de ADN , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Inestabilidad Genómica , Glioblastoma/genética , Glioblastoma/mortalidad , Glioblastoma/cirugía , Xenoinjertos , Humanos , Estimación de Kaplan-Meier , Ratones , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Pronóstico , Células Tumorales Cultivadas , Adulto JovenRESUMEN
Tumor-initiating cells are a subpopulation in aggressive cancers that exhibit traits shared with stem cells, including the ability to self-renew and differentiate, commonly referred to as stemness. In addition, such cells are resistant to chemo- and radiation therapy posing a therapeutic challenge. To uncover stemness-associated functions in glioma-initiating cells (GICs), transcriptome profiles were compared to neural stem cells (NSCs) and gene ontology analysis identified an enrichment of Ca2+ signaling genes in NSCs and the more stem-like (NSC-proximal) GICs. Functional analysis in a set of different GIC lines regarding sensitivity to disturbed homeostasis using A23187 and Thapsigargin, revealed that NSC-proximal GICs were more sensitive, corroborating the transcriptome data. Furthermore, Ca2+ drug sensitivity was reduced in GICs after differentiation, with most potent effect in the NSC-proximal GIC, supporting a stemness-associated Ca2+ sensitivity. NSCs and the NSC-proximal GIC line expressed a larger number of ion channels permeable to potassium, sodium and Ca2+. Conversely, a higher number of and higher expression levels of Ca2+ binding genes that may buffer Ca2+, were expressed in NSC-distal GICs. In particular, expression of the AMPA glutamate receptor subunit GRIA1, was found to associate with Ca2+ sensitive NSC-proximal GICs, and decreased as GICs differentiated along with reduced Ca2+ drug sensitivity. The correlation between high expression of Ca2+ channels (such as GRIA1) and sensitivity to Ca2+ drugs was confirmed in an additional nine novel GIC lines. Calcium drug sensitivity also correlated with expression of the NSC markers nestin (NES) and FABP7 (BLBP, brain lipid-binding protein) in this extended analysis. In summary, NSC-associated NES+/FABP7+/GRIA1+ GICs were selectively sensitive to disturbances in Ca2+ homeostasis, providing a potential target mechanism for eradication of an immature population of malignant cells.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias Encefálicas/tratamiento farmacológico , Calcio/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioma/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Células-Madre Neurales/efectos de los fármacos , Western Blotting , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Glioma/genética , Glioma/patología , Humanos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Transforming growth factor ß (TGFß) is a pluripotent cytokine promoting epithelial cell plasticity during morphogenesis and tumour progression. TGFß binding to type II and type I serine/threonine kinase receptors (TßRII and TßRI) causes activation of different intracellular signaling pathways. TßRI is associated with the ubiquitin ligase tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6). Here we show that TGFß, via TRAF6, causes Lys63-linked polyubiquitination of TßRI, promoting cleavage of TßRI by TNF-alpha converting enzyme (TACE), in a PKCζ-dependent manner. The liberated intracellular domain (ICD) of TßRI associates with the transcriptional regulator p300 to activate genes involved in tumour cell invasiveness, such as Snail and MMP2. Moreover, TGFß-induced invasion of cancer cells is TACE- and PKCζ- dependent and the TßRI ICD is localized in the nuclei of different kinds of tumour cells in tissue sections. Thus, our data reveal a specific role for TßRI in TGFß mediated tumour invasion.