Palbociclib Effectively Halts Proliferation but Fails to Induce Senescence in Patient-Derived Glioma Stem Cells.

Glioblastoma multiforme is the most aggressive primary brain tumor. Current knowledge suggests that the growth and recurrence of these tumors are due in part to the therapy-resistant glioma stem cell subpopulation, which possesses the ability for self-renewal and proliferation, driving tumor progression. In many cancers, the p16INK4a-CDK4/6-pRb pathway is disrupted in favor of cell cycle progression. In particular, the frequent deregulation of CDK4/6 in cancer positions these kinases as promising targets. Palbociclib, a potent and selective CDK4/6 inhibitor, has been approved by the FDA as a first-line treatment of advanced breast cancer and there is currently interest in evaluating its effect on other cancer types. Palbociclib has been reported to be efficient, not only at halting proliferation, but also at inducing senescence in different tumor types. In this study, we evaluated the effect of this inhibitor on four patient-derived glioma stem cell-enriched cell lines. We found that Palbociclib rapidly and effectively inhibits proliferation without affecting cell viability. We also established that in these cell lines CDK6 is the key interphase CDK for controlling cell cycle progression. Prolonged exposure to Palbociclib induced a senescent-like phenotype characterized by flattened morphology, cell cycle arrest, increased ß-galactosidase activity and induction of other senescent-associated markers. However, we found that after Palbociclib removal cell lines resumed normal proliferation, which implies they conserved their replicative potential. As a whole, our results indicate that in patient-derived glioma stem cell-enriched cell lines, Palbociclib induces a senescent-like quiescence rather than true senescence.

The Cell Cycle Inhibitors p21Cip1 and p27Kip1 Control Proliferation but Enhance DNA Damage Resistance of Glioma Stem Cells.

High-grade gliomas are the most prevalent and lethal primary brain tumors. They display a hierarchical arrangement with a population of self-renewing and highly tumorigenic cells called cancer stem cells. These cells are thought to be responsible for tumor recurrence, which make them main candidates for targeted therapies. Unbridled cell cycle progression may explain the selective sensitivity of some cancer cells to treatments. The members of the Cip/Kip family p21Cip1 and p27Kip1 were initially considered as tumor suppressors based on their ability to block proliferation. However, they are currently looked at as proteins with dual roles in cancer: one as tumor suppressor and the other as oncogene. Therefore, the aim of this study was to determine the functions of these cell cycle inhibitors in five patient-derived glioma stem cell-enriched cell lines. We found that these proteins are functional in glioma stem cells. They negatively regulate cell cycle progression both in unstressed conditions and in response to genotoxic stress. In addition, p27Kip1 is upregulated in nutrient-restricted and differentiating cells, suggesting that this Cip/Kip is a mediator of antimitogenic signals in glioma cells. Importantly, the lack of these proteins impairs cell cycle halt in response to genotoxic agents, rendering cells more vulnerable to DNA damage. For these reasons, these proteins may operate both as tumor suppressors, limiting cell proliferation, and as oncogenes, conferring cell resistance to DNA damage. Thus, deepening our knowledge on the biological functions of these Cip/Kips may shed light on how some cancer cells develop drug resistance.

Human Pluripotent Stem Cells and Derived Neuroprogenitors Display Differential Degrees of Susceptibility to BH3 Mimetics ABT-263, WEHI-539 and ABT-199.

Human embryonic stem cells (hESCs) are hypersensitive to genotoxic stress and display lower survival ability relative to their differentiated progeny. Herein, we attempted to investigate the source of this difference by comparing the DNA damage responses triggered by the topoisomerase I inhibitor camptothecin, in hESCs, human induced pluripotent stem cells (hiPSCs) and hESCs-derived neuroprogenitors (NP). We observed that upon camptothecin exposure pluripotent stem cells underwent apoptosis more swiftly and at a higher rate than differentiated cells. However, the cellular response encompassing ataxia-telangiectasia mutated kinase activation and p53 phosphorylation both on serine 15 as well as on serine 46 resulted very similar among the aforementioned cell types. Importantly, we observed that hESCs and hiPSCs express lower levels of the anti-apoptotic protein Bcl-2 than NP. To assess whether Bcl-2 abundance could account for this differential response we treated cells with ABT-263, WEHI-539 and ABT-199, small molecules that preferentially target the BH3-binding pocket of Bcl-xL and/or Bcl-2 and reduce their ability to sequester pro-apoptotic factors. We found that in the absence of stress stimuli, NP exhibited a higher sensitivity to ABT- 263 and WEHI-539 than hESCs and hiPSCs. Conversely, all tested cell types appeared to be highly resistant to the Bcl-2 specific inhibitor, ABT-199. However, in all cases we determined that ABT-263 or WEHI-539 treatment exacerbated camptothecin-induced apoptosis. Importantly, similar responses were observed after siRNA-mediated down-regulation of Bcl-xL or Bcl-2. Taken together, our results suggest that Bcl-xL contrary to Bcl-2 contributes to ensure cell survival and also functions as a primary suppressor of DNA double-strand brake induced apoptosis both in pluripotent and derived NP cells. The emerging knowledge of the relative dependence of pluripotent and progenitor cells on Bcl-2 and Bcl-xL activities may help to predict cellular responses and potentially manipulate these cells for therapeutic purposes in the near future.

Specific Preferences in Lineage Choice and Phenotypic Plasticity of Glioma Stem Cells Under BMP4 and Noggin Influence.

Although BMP4-induced differentiation of glioma stem cells (GSCs) is well recognized, details of the cellular responses triggered by this morphogen are still poorly defined. In this study, we established several GSC-enriched cell lines (GSC-ECLs) from high-grade gliomas. The expansion of these cells as adherent monolayers, and not as floating neurospheres, enabled a thorough study of the phenotypic changes that occurred during their differentiation. Herein, we evaluated GSC-ECLs' behavior toward differentiating conditions by depriving them of growth factors and/or by adding BMP4 at different concentrations. After analyzing cellular morphology, proliferation and lineage marker expression, we determined that GSC-ECLs have distinct preferences in lineage choice, where some of them showed an astrocyte fate commitment and others a neuronal one. We found that this election seems to be dictated by the expression pattern of BMP signaling components present in each GSC-ECL. Additionally, treatment of GSC-ECLs with the BMP antagonist, Noggin, also led to evident phenotypic changes. Interestingly, under certain conditions, some GSC-ECLs adopted an unexpected smooth muscle-like phenotype. As a whole, our findings illustrate the wide differentiation potential of GSCs, highlighting their molecular complexity and paving a way to facilitate personalized differentiating therapies.