Cytogenetic, genimic, epigenomic and drug sensitivity landscapes to unravel the complexity of glioma stem cell lines: a multi-level approach

BACKGROUND. Glioblastoma multiforme (GBM) is the most common and malignant type of glioma and it is characterized by extensive heterogeneity, both at the cellular and molecular level. The poor prognosis and the lack of an effective treatment are due to the presence of a small sub-population of cells with stem-like properties, termed glioma stem cells (GSCs). At the genomic level, heterogeneity is characterized by multiple levels of alterations, including cytogenetic, genomic and epigenomic alterations. Drug sensitivity is an additional level of GSC complexity and heterogeneity and the identification of an effective treatment for GBM depends on the depletion of the GSC pool. Valproic acid (VPA) is a histone deacetylase inhibitor and so it can be used for an epigenetic therapy for cancer. Besides, a differentiation inducing ability of VPA on cancer cells was demonstrated. Paclitaxel (PTX) is a conventional chemotherapeutic agent and in the last years it was shown to be a potential therapeutic drug for gliomas. AIMS AND PROJECT DESIGN. Six GSC lines were studied, as they represent a valuable tool for the investigation of cytogenomic and epigenomic landscapes of GBM, in order to unravel specific molecular pathways, involved in the stem-like counterpart. Drug sensitivity profiles were assessed, evaluating cell viability and cytomorphological parameters (mitotic index, ploidy and polymorphic nuclei), after VPA and PTX administration. The reliability of differentiation and epigenetic therapy through the use of VPA was further investigated by morphological and molecular epigenomic analysis, investigating the DNA methylation status. RESULTS AND DISCUSSION. Several shared cytogenetic and genomic alterations linked to GBM pathogenesis were found among the GSC lines. Specifically, polysomy of chromosome 7, loss of chromosome 10, CDKN2A and CDKN2B deletions are aberrations related to highly relevant pathways in GBM tumorigenesis. Moreover, a minimal deleted region at 1p36.31 was common among the six GSC lines, including CAMTA1 gene, a putative tumor suppressor gene, specific for cancer stem-like cells. Disregulated cytogenenomic pathways in GSCs were preferentially linked to the control of stem cell proliferation, invasion, cellular development and differentiation. The evaluation of the methylation profiles of GSC lines revealed aberrant methylation of developmental genes, which are targeted by Polycomb Repressive Complex 2 in embryonic stem cells and involved in cellular development and nervous system differentiation, evidencing a specific impairment of these processes in cancer stem-like cells. VPA is able to begin a differentiation process in GSCs, as demonstrated by the study of methylation changes caused by VPA, through the methylation of pathways which are involved in self-renewal maintenance, such as Wnt/β-catenin, and several cancer-related mechanisms. Anyway, terminal differentiation was impaired, due to an intrinsic characteristic of cancer cells endowed with stem like properties. GSCs viability was severely affected by dual drug treatment, combining VPA and PTX: VPA caused an initial differentiation, enabling PTX to induce cell death of downstream cells in tumor hierarchy. Thus, a dual approach with drugs affecting different features of malignancy could be a successful approach to GBM treatment. CONCLUSIONS. A multi-level study for the evaluation of cytogenomic and epigenomic landscapes of GSCs is an effective approach for the identification of molecular pathways, specifically de-regulated in stem-like cells, giving an outstanding contribution in the identification of key mechanisms sustaining self-renewal. GSC lines are a valuable tool to evaluate the potentiality of new therapeutical approaches, which should be able to overwhelm the stem-like related counterpart. VPA and PTX combined treatment was found to fulfill the therapeutical potential of VPA and might be a successful approach to unlock the self-renewal loop, typical of GSCs and affect their growth.