Ion channel expression patterns in glioblastoma stem cells with functional and therapeutic implications for malignancy

<div><p>Ion channels and transporters have increasingly recognized roles in cancer progression through the regulation of cell proliferation, migration, and death. Glioblastoma stem-like cells (GSCs) are a source of tumor formation and recurrence in glioblastoma multiforme, a highly aggressive brain cancer, suggesting that ion channel expression may be perturbed in this population. However, little is known about the expression and functional relevance of ion channels that may contribute to GSC malignancy. Using RNA sequencing, we assessed the enrichment of ion channels in GSC isolates and non-tumor neural cell types. We identified a unique set of GSC-enriched ion channels using differential expression analysis that is also associated with distinct gene mutation signatures. In support of potential clinical relevance, expression of selected GSC-enriched ion channels evaluated in human glioblastoma databases of The Cancer Genome Atlas and Ivy Glioblastoma Atlas Project correlated with patient survival times. Finally, genetic knockdown as well as pharmacological inhibition of individual or classes of GSC-enriched ion channels constrained growth of GSCs compared to normal neural stem cells. This first-in-kind global examination characterizes ion channels enriched in GSCs and explores their potential clinical relevance to glioblastoma molecular subtypes, gene mutations, survival outcomes, regional tumor expression, and experimental responses to loss-of-function. Together, the data support the potential biological and therapeutic impact of ion channels on GSC malignancy and provide strong rationale for further examination of their mechanistic and therapeutic importance.</p></div

Fragment Length of Circulating Tumor DNA

<div><p>Malignant tumors shed DNA into the circulation. The transient half-life of circulating tumor DNA (ctDNA) may afford the opportunity to diagnose, monitor recurrence, and evaluate response to therapy solely through a non-invasive blood draw. However, detecting ctDNA against the normally occurring background of cell-free DNA derived from healthy cells has proven challenging, particularly in non-metastatic solid tumors. In this study, distinct differences in fragment length size between ctDNAs and normal cell-free DNA are defined. Human ctDNA in rat plasma derived from human glioblastoma multiforme stem-like cells in the rat brain and human hepatocellular carcinoma in the rat flank were found to have a shorter principal fragment length than the background rat cell-free DNA (134–144 bp vs. 167 bp, respectively). Subsequently, a similar shift in the fragment length of ctDNA in humans with melanoma and lung cancer was identified compared to healthy controls. Comparison of fragment lengths from cell-free DNA between a melanoma patient and healthy controls found that the <i>BRAF</i> V600E mutant allele occurred more commonly at a shorter fragment length than the fragment length of the wild-type allele (132–145 bp vs. 165 bp, respectively). Moreover, size-selecting for shorter cell-free DNA fragment lengths substantially increased the <i>EGFR</i> T790M mutant allele frequency in human lung cancer. These findings provide compelling evidence that experimental or bioinformatic isolation of a specific subset of fragment lengths from cell-free DNA may improve detection of ctDNA.</p></div

Ion channel blockade reduces GSC viability.

<p>A. Summary of functionally-related ion channel families enriched in GSCs (orange) compared to controls. Y-axis, mean log₂ fold change (GSC vs. NSC/NHA) values for each ion channel family; x-axis, proportion of ion channel family members > 2-fold change (GSC vs. NSC/NHA); bubble size corresponds to average GSC CPM value. Genes were excluded if average NSC/NHA values were zero. Families were considered if they contained more than one member. B. Proportion of ion channel family members expressed at >1 FPKM within distinct GBM regional compartments as revealed by Ivy GAP analysis (only families with at least three members are shown). C. Antagonists for top IGC families and selected specific ion channel blockers were applied to GSC-0827, GSC-0131, and NSC-CB660 in media at indicated concentrations. MTT viability assay was performed at 72 hours. Fluorescence arbitrary units (AU) were averaged across triplicates and normalized to control media conditions (dashed line at 1.0). Bars, mean ± SEM. N = 3–4. Repeated measures two-way ANOVA with Dunnett’s multiple comparison test compared to NSC-CB660. n.s., not significant.</p

IGCs are enriched in distinct GBM tumor regions.

<p>A. Heat map of expression levels of the top 25 IGCs in various GBM tumor regions from the Ivy GAP RNA-seq database. B. IGCs associated with particular molecular subtypes in CT samples. Colored headers indicate predominant subtype associated with that ion channel. Bars, mean ± SEM. Kruskal-Wallis test across molecular subtypes. C. Survival curve for <i>CNGA3</i> within CT samples stratified high/low by median FPKM value (3.908). Log-rank (Mantel-Cox) test. Median days to death for each group reported on graph. Leading Edge (LE), Infiltrating Tumor (IT), Cellular Tumor (CT), Perinecrotic zone (PZ), Pseudopalisading cells around necrosis (PAN), Hyperplastic blood vessels in cellular tumor (HBV), Microvascular Proliferation (MVP).</p

GSC-enriched ion channels are associated with GBM molecular features.

<p>A. Hierarchical clustering of GSCs by molecular subtype based on CPM values of all 266 ion channels. B. Correlation of IGCs with gene mutation signatures of critical GBM signaling pathways (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172884#pone.0172884.s001" target="_blank">S1 Table</a> for details). Bars, mean ± min/max, IQR. Two-tailed Mann-Whitney test; *p<0.05, **p<0.01.</p

RNA sequencing identifies ion channels enriched in GSCs.

<p>A. Mean GSC CPM values plotted against mean control (NSC and NHA) CPM values for all ion channel-related genes. Red and blue points represent genes that are differentially expressed ≥|2| log₂ fold change in each subclass. B. Heat map of the most differentially enriched ion channels in GSCs compared to control NSCs and NHAs by GSEA analysis. Each column represents log₂ fold-change values (compared to averaged values across NSCs/NHAs) from a distinct cell isolate after averaging triplicate CPM values. Ion channels for which average GSC CPM values were <1 were not included, and individual CPM values of 0 were replaced with 0.01. C. CPM values for six of the most differentially enriched genes shown in panel (B). Bars, mean ± SEM. Mann-Whitney test; *p<0.05, **p<0.01, ns = not significant. D. Real-time qPCR analysis of a selected number of IGCs in several GSC isolates. C_T values were normalized to <i>ACTB</i> (β-Actin) C_T values; ddC_T values relative to NSC-CB660 are shown. Bars, mean ± stdev. N = 3. GSC, Glioblastoma stem-like cells; NSC, human fetal neural stem cells including c-myc immortalized from cortex (NSC-CX) and brainstem (NSC-VM); NHA, normal human astrocytes including RasV12 transformed (NHA-RAS).</p

Selection of shorter cell-free DNA fragments enriched for ctDNA.

<p>In <b>A-D</b>, cell-free DNA fragment size distribution by densitometry and the corresponding digital droplet PCR results for mutant allele frequency are shown for each tumor patient. In <b>A-C</b>, the first column identifies the fragment size distribution for the fraction associated with the largest increase in mutant allele frequency (red or purple curve) along with the distribution of the corresponding library (black curve). In <b>D</b>, the first column shows a similar fraction for LC1 as presented in <b>A-C</b>. In <b>A-D</b>, the color of each curve matches the gel location as depicted in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006162#pgen.1006162.s008" target="_blank">S8 Fig</a>. In <b>A-D</b>, the middle and last columns report the digital droplet PCR results for mutant allele frequency (%) in the library and the gel fraction, respectively. In <b>E</b>, the ratio of the mutant allele frequency in each fraction to the MAF in the library was plotted for each tumor patient. The dashed gray line represents a ratio of 1 (i.e., no increase or decrease in MAF). To account for variability during gel fraction excision between samples, the <i>x</i>-axis location for plotting values associated with each gel fraction was determined via densitometry by subtracting the peak fragment length for each library from the peak fragment length for each fraction. Negative values correspond to shorter fragments and positive values correspond to longer fragments. The blue shaded box identifies the region where increase in the mutant allele frequency was the greatest across all samples.</p

The cell-free DNA and ctDNA from melanoma patients consisted of shorter fragments than the healthy controls and the WT allele fragments.

<p>In <b>A</b>, the relative fragment length of cell-free DNA obtained from melanoma patients with and without metastatic disease (± lymph node, LN; <b>A</b>) tended to be shorter by densitometry compared to cell-free DNA from four healthy controls (<b>A</b>, gray lines). In <b>B</b>, the fragment lengths derived from cell-free DNA deep-sequencing in a patient with melanoma (<b>A</b>, black arrow) were generally shorter than the fragment lengths present in the pool of healthy controls (green and purple lines, respectively). (<b>C)</b> In the melanoma patient, cell-free DNA fragment lengths containing the mutant allele (<i>BRAF</i> V600E, red line) were shorter compared to the fragment lengths containing the wild-type (WT) allele (blue line). In the shorter fragments there was general overlap between the mutant and WT allele sizes since the <i>BRAF</i> V600E mutation is heterozygous. <b>(D)</b> Fragment lengths between 110–140 bp had the highest proportion of the mutant allele (<b>D</b>, red solid line; the mutant allele frequency <100 bp was erratic due to few observations). In <b>D</b>, the solid black line represents the overall frequency for each range of fragment lengths in the melanoma patient and indicates that there may be insufficient amount of DNA for detecting mutant alleles below 100 bp. Of note, the WT allele from the healthy control occurred more commonly between 160–180 bp (<b>D</b>, blue dashed line).</p

IGCs are associated with poor clinical outcomes.

<p>Expression levels of IGCs were identified from 525 TCGA bulk GBM microarray expression samples. Samples for which expression levels were highest and lowest (top and bottom 10%) were then compared for time to death. Median days to death for each group reported on graph.</p

siRNA-mediated knockdown of selected IGCs impairs GSC viability.

<p>A. Real-time qPCR analysis of IGC expression levels 24 hours after transfection of scrambled negative control siRNA (siScr) and individual siRNA candidates (1, 2, 3) for each IGC. C_T values were normalized to <i>ACTB</i> (β-Actin) C_T values; ddC_T from siScr. Bars, mean ± stdev. N = 3. One-way ANOVA with Dunnet’s post-test compared to siScr, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. B. CellTiter-Glo viability assay 72 hours following siRNA-mediated knockdown of IGCs. GSC viability decreased in response to increasing amounts of siRNA (2, 5, and 20 pmol). Fluorescence arbitrary units (AU) were normalized to siScr levels. Bars, mean ± SEM. N = 3–4. Two-way ANOVA with repeated measures; <i>SCN8A</i>: cell type p = 0.0001, dose p<0.0001, interaction p<0.0001; <i>KCNB1</i>: cell type p = 0.0042, dose p<0.0001, interaction p<0.0001; <i>GRIA3</i>: cell type p = 0.0011, dose p<0.0001, interaction p<0.0001.</p