Identification of compounds that target glioma initiating cells

Glioblastoma multiforme is a common form of brain tumor that leads to debilitating effects despite the current regiment of treatment, which includes surgery, chemotherapy and radiotherapy. With the discovery of glioma-initiating cells (GICs) that exists within the bulk tumor, some light has been shed on this disease. GICs have been shown to be resistant to chemotherapy and radiotherapy, therefore providing a plausible explanation for the high propensity for recurrence in patients. However, effective treatments are still unavailable, and there is an urgency to discover drugs that can eradicate this group of cells. The overall aim of this thesis is to identify drugs from small molecule screens that can kill GICs, as well as to understand the possible causes and mechanisms for drug sensitivity. In Paper I, we have identified a new small molecule, Vacquinol-1, that reduced the viability and growth of GICs, both in vitro and in vivo, and had little effects on normal cells such as fibroblasts and embryonic stem cells. Cell death was caused by an unconventional nonapoptotic manner, where the cells showed massive accumulation of vacuoles that formed through macropinocytosis. This led to the impairment of cell function followed by cell death. Furthermore, treatment with Vacquinol-1 also exhibited excellent improvement in survival of glioma xenografted mice. Using an shRNA screen, mitogen-activated protein kinase kinase 4 (MKK4), which is involved in stress response, was determined to play a role in the unique type of cell death. In Paper II, we have identified that GICs are particularly sensitive to perturbations in calcium (Ca2+) homeostasis, with the relative degree of sensitivity being linked to their degree of stemness. The two compounds employed in this study, Ca2+ ionophore A23187 and the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitor Thapsigargin, are wellknown compounds that elicit cell death. However, the compounds affected viability to a different degree in the various GIC lines, with the line that is more similar to neural stem cells being more sensitive to perturbations in Ca2+ homeostasis. This sensitivity was correlated to expression levels of different Ca2+ related proteins such as GRIA1 and S100A6, as well as to Nestin (NES). In Paper III, we have repositioned Niguldipine, an old anti-hypertensive drug, as a potential compound for targeting GICs. Through an ion channel drug screen, we have also observed that GICs exhibit sensitivity to Ca2+ modulators, suggesting once again that Ca2+ homeostasis is critical to cell viability in GICs. Niguldipine, which is also a Ca2+ channel inhibitor, also showed a selection for GICs as compared to normal cells such as fibroblasts and neural stem cells. At the effective dose, the compound showed no effects on cardiac rhythmicity, and administration of the drug resulted in a significant improvement in the survival of glioma xenografted mice. In conclusion, we have identified a few small molecules, both old and new, that can reduce the proliferation and viability of GICs without affecting that of normal cells. One of the mechanisms underlying selectivity is that GICs show a greater degree of sensitivity to disturbances in Ca2+ homeostasis, therefore suggesting that Ca2+ modulators should be screened for their potential in cancer therapy

Прикладна механіка і основи конструювання: навчально-методичний посібник

Розроблено відповідно до навчальної програми і призначено для виконання розрахунково-графічної роботи з дисципліни «Прикладна механіка і основи конструювання» студентам напряму підготовки 6.050202 «Автоматизація та компютерно-ігрегровані технології» денної та заочної форм навчання. Посібник рекомендовано також для самостійної роботи студентів, оскільки він вміщує короткі теоретичні викладки основного матеріалу дисципліни «Прикладна механіка і основи конструювання», умови завдань, приклади їх розв’язування, необхідні довідкові дані

Selective Calcium Sensitivity in Immature Glioma Cancer Stem Cells

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

Expression of genes involved in Ca²⁺ signaling in GICs correlating with a NSC-associated transcriptome.

<p>(A) GIC lines rank ordered in relation to NSC lines (second component in a principle component analysis of microarray based mRNA expression data from Pollard et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115698#pone.0115698-Pollard1" target="_blank">[11]</a>, where the first component segregates NSCs and GICs from normal brain tissue). GliNS1 is derived from the G144ED line in the Pollard et al study. (B) Re-analysis of transcriptome profiles in Pollard et al comparing GICs to NSCs indicating a NSC-proximal cluster of stem-like GICs with high similarity to NSCs, sharing e.g. SOX2 and BLBP expression. NSC-distal GIC lines in contrast expressed microglia markers, such as CXCL2, CXCL5 and CCL20. (C) De novo RNA sequencing analysis and pairwise comparisons of NSCs and three individual GIC lines (GliNS1, G179NS and G166NS) showed that NSCs expressed a larger number of genes with 10-fold higher gene expression compared to all GIC lines. (D) Pairwise comparisons of NSCs to the GIC lines GliNS1, G179NS and G166NS, individually. Gene enrichment and gene ontology analysis of sequencing based transcriptome profiles, identified an enrichment of Ca²⁺ signaling genes in NSCs, which increased with rank order distal to NSC in pairwise comparisons. (E) Pairwise comparisons of the NSC-proximal (GliNS1) and NSC-distal (G166NS) GICs. Gene enrichment and gene ontology analysis suggested a switch in Ca²⁺ permeable channels to Ca²⁺ binding genes in the NSC-distal GIC line (upper boxes). In volcano plot, gene names in green denote ion channel/pump/transporter related genes, whereas gene names in purple denote Ca²⁺ binding proteins genes. The volcano plot of the comparison of NSC-proximal and NSC-distal GICs revealed a larger number of ion channels expressed in the NSC-proximal GIC (GliNS1).</p

Transcriptome analysis of drug response in GliNS1 and G166NS.

<p>Transcriptional response to increased cytosolic Ca²⁺ (A23187), was investigated by RNA sequencing after 7 hours of drug exposure in the NSC-proximal GIC line GliiNS1 and the NSC-distal line G166NS. Volcano plots of significantly (p<0.05) altered gene expression in GliNS1 (A) and G166NS (C) with shared induced genes marked in red and green (Ca²⁺ activated transcription factor NFATC2). Note the differences in x-axis indicating higher all global induction of gene expression in GliNS1. (B) Gene enrichment and gene ontology analysis of genes with a significant change in expression (p<0.05) in GliNS1, identified genes involved in cell cycle progression as well as ER/golgi associated functions and cellular stress response. (D) Gene enrichment analysis of genes downregulated at least 3-fold in GliNS1 and upregulated at least 1.5-fold in G166NS.</p

Gene expression correlating with high Ca²⁺ sensitivity in 9 GIC lines.

<p>(A) A correlation analysis of genome wide mRNA expression (microarray analysis) and sensitivity to Thapsigargin (1 uM) in 9 additional GIC lines, retrieved 785 genes correlating with Ca²⁺ drug sensitivity. Gene enrichment and ontology analyses identified involvement of genes affecting proliferation, oxygen and RNA metabolism, catabolism and Ca²⁺-mediated signaling. (B) 385 genes positively correlating with high sensitivity were filtered first for genes also expressed higher in the NSC-proximal GIC line GliNS1 and thereafter also being downregulated in this line upon differentiation, which was found to reduce Ca²⁺ drug sensitivity, retrieving a set of nine genes, including the AMPA receptor coding GRIA1.</p

Membrane-depolarizing channel blockers induce selective glioma cell death by impairing nutrient transport and unfolded protein/amino acid responses.

Glioma-initiating cells (GIC) are considered the underlying cause of recurrences of aggressive glioblastomas, replenishing the tumor population and undermining the efficacy of conventional chemotherapy. Here we report the discovery that inhibiting T-type voltage-gated Ca2+ and KCa channels can effectively induce selective cell death of GIC and increase host survival in an orthotopic mouse model of human glioma. At present, the precise cellular pathways affected by the drugs affecting these channels are unknown. However, using cell-based assays and integrated proteomics, phosphoproteomics, and transcriptomics analyses, we identified the downstreamsignaling events these drugs affect. Changes in plasma membrane depolarization and elevated intracellular Na+, which compromised Na+-dependent nutrient transport, were documented. Deficits in nutrient deficit acted in turn to trigger the unfolded protein response and the amino acid response, leading ultimately to nutrient starvation and GIC cell death. Our results suggest new therapeutic targets to attack aggressive gliomas