Concurrent Activation of Both Survival-Promoting and Death-Inducing Signaling by Chloroquine in Glioblastoma Stem Cells : Implications for Potential Risks and Benefits of Using Chloroquine as Radiosensitizer

Lysosomotropic agent chloroquine was shown to sensitize non-stem glioblastoma cells to radiation in vitro with p53-dependent apoptosis implicated as one of the underlying mechanisms. The in vivo outcomes of chloroquine or its effects on glioblastoma stem cells have not been previously addressed. This study undertakes a combinatorial approach encompassing in vitro, in vivo and in silico investigations to address the relationship between chloroquine-mediated radiosensitization and p53 status in glioblastoma stem cells. Our findings reveal that chloroquine elicits antagonistic impacts on signaling pathways involved in the regulation of cell fate via both transcription-dependent and transcription-independent mechanisms. Evidence is provided that transcriptional impacts of chloroquine are primarily determined by p53 with chloroquine-mediated activation of pro-survival mevalonate and p21-DREAM pathways being the dominant response in the background of wild type p53. Non-transcriptional effects of chloroquine are conserved and converge on key cell fate regulators ATM, HIPK2 and AKT in glioblastoma stem cells irrespective of their p53 status. Our findings indicate that pro-survival responses elicited by chloroquine predominate in the context of wild type p53 and are diminished in cells with transcriptionally impaired p53. We conclude that p53 is an important determinant of the balance between pro-survival and pro-death impacts of chloroquine and propose that p53 functional status should be taken into consideration when evaluating the efficacy of glioblastoma radiosensitization by chloroquine

Diversity of Clinically Relevant Outcomes Resulting from Hypofractionated Radiation in Human Glioma Stem Cells Mirrors Distinct Patterns of Transcriptomic Changes

Hypofractionated radiotherapy is the mainstay of the current treatment for glioblastoma. However, the efficacy of radiotherapy is hindered by the high degree of radioresistance associated with glioma stem cells comprising a heterogeneous compartment of cell lineages differing in their phenotypic characteristics, molecular signatures, and biological responses to external signals. Reconstruction of radiation responses in glioma stem cells is necessary for understanding the biological and molecular determinants of glioblastoma radioresistance. To date, there is a paucity of information on the longitudinal outcomes of hypofractionated radiation in glioma stem cells. This study addresses long-term outcomes of hypofractionated radiation in human glioma stem cells by using a combinatorial approach integrating parallel assessments of the tumor-propagating capacity, stemness-associated properties, and array-based profiling of gene expression. The study reveals a broad spectrum of changes in the tumor-propagating capacity of glioma stem cells after radiation and finds association with proliferative changes at the onset of differentiation. Evidence is provided that parallel transcriptomic patterns and a cumulative impact of pathways involved in the regulation of apoptosis, neural differentiation, and cell proliferation underly similarities in tumorigenicity changes after radiation

GARP as an immune regulatory molecule in the tumor microenvironment of glioblastoma multiforme

Glycoprotein A repetition predominant (GARP), a specific surface molecule of activated regulatory T cells, has been demonstrated to significantly contribute to tolerance in humans by induction of peripheral Treg and regulatory M2-macrophages and by inhibition of (tumorantigen specific) T effector cells. Previous work identified GARP on Treg, and also GARP on the surface of several malignant tumors, as well as in a soluble form being shedded from their surface, contributing to tumor immune escape. Preliminary results also showed GARP expression on brain metastases of malignant melanoma. On the basis of these findings, we investigated whether GARP is also expressed on primary brain tumors. We showed GARP expression on glioblastoma (GB) cell lines and primary GB tissue, as well as on low-grade glioma, suggesting an important influence on the tumor micromilieu and the regulation of immune responses also in primary cerebral tumors. This was supported by the finding that GB cells led to a reduced, in part GARP-dependent effector T cell function (reduced proliferation and reduced cytokine secretion) in coculture experiments. Interestingly, GARP was localized not only on the cell surface but also in the cytoplasmatic, as well as nuclear compartments in tumor cells. Our findings reveal that GARP, as an immunoregulatory molecule, is located on, as well as in, tumor cells of GB and low-grade glioma, inhibiting effector T cell function, and thus contributing to the immunosuppressive tumor microenvironment of primary brain tumors. As GARP is expressed on activated Treg, as well as on brain tumors, it may be an interesting target for new immunotherapeutic approaches using antibody-based strategies as this indication

ALDH1A3 Segregated Expression and Nucleus-Associated Proteasomal Degradation Are Common Traits of Glioblastoma Stem Cells

Aldehyde dehydrogenase 1 isoforms A1 and A3 have been implicated as functional biomarkers associated with distinct molecular subtypes of glioblastoma and glioblastoma stem cells. However, the exact roles of these isoforms in different types of glioma cells remain unclear. The purpose of this study was to dissect the association of A1 or A3 isoforms with stem and non-stem glioblastoma cells. This study has undertaken a systematic characterization of A1 and A3 proteins in glioblastoma tissues and a panel of glioblastoma stem cells using immunocytochemical and immunofluorescence staining, Western blot and the subcellular fractionation methodology. Our main findings are (i) human GSCs express uniformly ALDH1A3 but not the ALDH1A1 isoform whereas non-stem glioma cells comparably express both isoforms; (ii) there is an abundance of ALDH1A3 peptides that prevail over the full-length form in glioblastoma stem cells but not in non-stem glioma cells; (iii) full-length ALDH1A3 and ALDH1A3 peptides are spatially segregated within the cell; and (vi) the abundance of full-length ALDH1A3 and ALDH1A3 peptides is sensitive to MG132-mediated proteasomal inhibition. Our study further supports the association of ALDH1A3 with glioblastoma stem cells and provide evidence for the regulation of ALDH1A3 activities at the level of protein turnover

Concurrent Activation of Both Survival-Promoting and Death-Inducing Signaling by Chloroquine in Glioblastoma Stem Cells: Implications for Potential Risks and Benefits of Using Chloroquine as Radiosensitizer

Lysosomotropic agent chloroquine was shown to sensitize non-stem glioblastoma cells to radiation in vitro with p53-dependent apoptosis implicated as one of the underlying mechanisms. The in vivo outcomes of chloroquine or its effects on glioblastoma stem cells have not been previously addressed. This study undertakes a combinatorial approach encompassing in vitro, in vivo and in silico investigations to address the relationship between chloroquine-mediated radiosensitization and p53 status in glioblastoma stem cells. Our findings reveal that chloroquine elicits antagonistic impacts on signaling pathways involved in the regulation of cell fate via both transcription-dependent and transcription-independent mechanisms. Evidence is provided that transcriptional impacts of chloroquine are primarily determined by p53 with chloroquine-mediated activation of pro-survival mevalonate and p21-DREAM pathways being the dominant response in the background of wild type p53. Non-transcriptional effects of chloroquine are conserved and converge on key cell fate regulators ATM, HIPK2 and AKT in glioblastoma stem cells irrespective of their p53 status. Our findings indicate that pro-survival responses elicited by chloroquine predominate in the context of wild type p53 and are diminished in cells with transcriptionally impaired p53. We conclude that p53 is an important determinant of the balance between pro-survival and pro-death impacts of chloroquine and propose that p53 functional status should be taken into consideration when evaluating the efficacy of glioblastoma radiosensitization by chloroquine

CD133 Expression Is Not Synonymous to Immunoreactivity for AC133 and Fluctuates throughout the Cell Cycle in Glioma Stem-Like Cells

<div><p>A transmembrane protein CD133 has been implicated as a marker of stem-like glioma cells and predictor for therapeutic response in malignant brain tumours. CD133 expression is commonly evaluated by using antibodies specific for the AC133 epitope located in one of the extracellular domains of membrane-bound CD133. There is conflicting evidence regarding the significance of the AC133 epitope as a marker for identifying stem-like glioma cells and predicting the degree of malignancy in glioma cells. The reasons for discrepant results between different studies addressing the role of CD133/AC133 in gliomas are unclear. A possible source for controversies about CD133/AC133 is the widespread assumption that expression patterns of the AC133 epitope reflect linearly those of the CD133 protein. Consequently, the readouts from AC133 assessments are often interpreted in terms of the CD133 protein. The purpose of this study is to determine whether and to what extent do the readouts obtained with anti-AC133 antibody correspond to the level of CD133 protein expressed in stem-like glioma cells. Our study reveals for the first time that CD133 expressed on the surface of glioma cells is poorly immunoreactive for AC133. Furthermore, we provide evidence that the level of CD133 occupancy on the surface of glioma cells fluctuates during the cell cycle. Our results offer a new explanation for numerous inconsistencies regarding the biological and clinical significance of CD133/AC133 in human gliomas and call for caution in interpreting the lack or presence of AC133 epitope in glioma cells.</p></div

Comparative analysis of the total and membrane-associated CD133 protein in CaCo-2 cells.

<p>A. Western blot analysis of crude lysates (100 μg total protein per lane) from mock-treated CaCo-2 cells (lanes 1) or CaCo-2 cells transfected with the validated anti-CD133 siRNA (LifeTechnologies) (lanes 2–4). After the transfer to the PVDF membrane, Ponceau S staining was done to verify the efficacy of protein transfer (<i>top panel</i>) and followed by sequential probing with anti-AC133 Ab (<i>middle panel</i>) and anti-CD133CT Ab (<i>bottom panel</i>), respectively. B. Comparative assessment of CD133 in unfractionated cell lysates (“input”, 20 μg) and plasma membranes (“PM”, 1 μg) by anti-AC133 or anti-CD133CT Abs.</p

Comparative assessment of the AC133 epitope and CD133 protein in human GCSs.

<p>A. Representative histograms showing CD133 surface expression detected by anti-AC133 Ab (<i>left panels</i>) or anti-CD133CT Ab (<i>right panels</i>) in primary GSCs cultures and stem-like glioma clone G112SP. B. Mean percentage of cells positively labelled with anti-AC133 Ab (<i>blue</i>) or anti-CD133CT Ab (<i>black)</i> in a panel of GSC lines and CaCo-2 cells used as a positive control. C. Comparative assessment of the total (“input”) and membrane-associated (“PM”) CD133 protein in human GSC lines No. 1063, No. 1080 and No. 1051. 20 μg of proteins were loaded per lane. D. Representative histograms showing surface expression of CD133/2 detected by anti-AC141 Ab in primary cultures of GSCs, stem-like glioma clone G112SP and reference cell line CaCo-2 used as a positive control.</p

Divergent patterns detected by anti-AC133 and anti-CD133CT Abs in CaCo-2 cells.

<p>A. Representative flow cytometry histograms of CaCo-2 cells immunolabeled with anti-AC133 Ab (<i>left panel</i>) or anti-CD133CT Ab (<i>right panel</i>). B. Quantification of AC133- and CD133CT-positive cells from three independent experiments. Bars represent mean percentage of positive cells ± SEM. C. Immunofluorescence microscopy of CaCo-2 cells co-stained with anti-AC133 Ab (green) and anti-CD133CT Ab (red). Nuclei were counterstained using DAPI (blue). Confocal microscopy images (<i>inset</i>) show the subcellular distribution of the staining. Merged image shows overlapping signals generated by anti-AC133 Ab (<i>green</i>) and anti-CD133CT Ab (<i>red</i>) antibodies. Scale bars correspond to 50 μm (main fields) and 7 μm (insets).</p

Comparative assessment of surface CD133 in unsynchronized and synchronized GSCs.

<p>A. Thymidine treatment scheme used to synchronize human GSCs. B. Representative histograms of No. 1051 GSCs labeled in parallel with propidium iodide (PI, <i>top row</i>) and anti-CD133CT Ab (<i>bottom row</i>). Fluorescence intensity is represented as arbitrary units (AU) in a linear (PI, <i>top row</i>) or logarithmic (anti-CD133CT Ab, <i>bottom row</i>) scale. C. Quantitative results of three independent experiments. Shown is the percentage of cells positively labeled with anti-CD133CT Ab in untreated (“control”) or thymidine treated GSCs. GSCs were stained either immediately after the second thymidine treatment (“2xThy”) or after a twelve hours release from a double thymidine block (“2xThy + Release”).</p