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Supplementary Figure 3 from Inhibition of the Sodium-Potassium-Chloride Cotransporter Isoform-1 Reduces Glioma Invasion

Supplementary Methods, Figure Legends 1-3 from Inhibition of the Sodium-Potassium-Chloride Cotransporter Isoform-1 Reduces Glioma Invasion

Supplementary Figure 1 from Inhibition of the Sodium-Potassium-Chloride Cotransporter Isoform-1 Reduces Glioma Invasion

Supplementary Figure 2 from Inhibition of the Sodium-Potassium-Chloride Cotransporter Isoform-1 Reduces Glioma Invasion

Malignant gliomas metastasize throughout the brain by infiltrative cell migration into peritumoral areas. Invading cells undergo profound changes in cell shape and volume as they navigate extracellular spaces along blood vessels and white matter tracts. Volume changes are aided by the concerted release of osmotically active ions, most notably K+ and Cl−. Their efflux through ion channels along with obligated water causes rapid cell shrinkage. Suitable ionic gradients must be established and maintained through the activity of ion transport systems. Here, we show that the Sodium-Potassium-Chloride Cotransporter Isoform-1 (NKCC1) provides the major pathway for Cl− accumulation in glioma cells. NKCC1 localizes to the leading edge of invading processes, and pharmacologic inhibition using the loop diuretic bumetanide inhibits in vitro Transwell migration by 25% to 50%. Short hairpin RNA knockdowns of NKCC1 yielded a similar inhibition and a loss of bumetanide-sensitive cell volume regulation. A loss of NKCC1 function did not affect cell motility in two-dimensional assays lacking spatial constraints but manifested only when cells had to undergo volume changes during migration. Intracranial implantation of human gliomas into severe combined immunodeficient mice showed a marked reduction in cell invasion when NKCC1 function was disrupted genetically or by twice daily injection of the Food and Drug Administration–approved NKCC1 inhibitor Bumex. These data support the consideration of Bumex as adjuvant therapy for patients with high-grade gliomas. Cancer Res; 70(13); 5597–606. ©2010 AACR.

Gliomas account for the majority of primary brain neoplasias and are among the most lethal human tumors. Tumor cells diffusely infiltrate surrounding brain tissue. The invasion of glioma cells into normal brain is thought to be facilitated by ion channels which dynamically regulate cell volume and the ability of tumor cells to shrink and invade through tortuous extracellular spaces in the brain. To shrink, glioma cells release K+ and Cl- through ion channels, which induce water to leave the cells through aquaporins. Recent evidence suggests that CL- transport in glioma cells is mediated primarily by the voltage-gated chloride channel family members, CLCN2 and CLCN3, making them attractive candidate genes in glioma pathogenesis. In the present study, we tested whether single nucleotide polymorphisms (SNPs) in CLCN2 (3q27) and CLCN3 (4q33) are associated with glioma risk or patient outcome in a clinic-based case-control study conducted at medical centers in the southeastern US. A total of 15 candidate and haplotype tagging SNPs (4 SNPs in CLCN2 and 11 SNPs in CLCN3) were genotyped using the Illumina Goldengate assay in 563 newly diagnosed (eg. nonrecurrent) glioma cases (including 324 WHO grade IV glioblastomas (GBM); 145 WHO grade II or III astrocytomas and 94 oligoastrocytomas and oligodendrogliomas) and 629 healthy controls. DNA was isolated from saliva samples. Logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CI) for glioma risk in relation to individual genotypes adjusting for age and gender. Proportional hazards regression was used to estimate age and gender-adjusted hazard ratios (HR) for GBM-related death (203 deaths; median follow up: 11.2 months) for each SNP. No consistent associations were observed for any examined variant in CLCN2. In contrast, 6 of the examined SNPs in CLCN3, all in moderate linkage disequilibrium (r2: 0.30-0.80), were significantly associated with glioma risk. Among them, the strongest signal was observed in rs3797040 (NT_016354.19:g.95088578C>T), a SNP located in a putative transcription binding site (per variant “T” allele OR: 1.25; 95% CI:1.05-1.47; p=0.0095; allele frequency: 0.49); the observed excess risk associated with the variant rs3797040 allele was consistent in GBM (per allele OR: 1.23; 95% CI: 1.01-1.51; p=0.042), astrocytomas (per allele OR: 1.15; 95% CI: 0.88-1.49; p=0.312) and oligodendrogliomas (per allele OR: 1.75; 95% CI: 1.23-2.44; p=0.001). The ancestral “C” allele in CLCN3 rs3797040 was associated with increased mortality rates among the patients with GBM (per allele HR: 1.31; 95% CI: 1.06-1.62; p=0.011). To our knowledge this is the first report suggesting that genetic variation in plasma membrane ion channels may be a determinant of glioma risk. Further studies are needed to confirm these observations and identify the putative causal variant. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 876. doi:10.1158/1538-7445.AM2011-876