Disruption of transient receptor potential canonical channel 1 causes incomplete cytokinesis and slows the growth of human malignant gliomas

Abstract

Despite decades of research, primary brain tumors, gliomas, lack effective treatment options and present a huge clinical challenge. Particularly, the most malignant subtype, Glioblastoma multiforme, proliferates extensively and cells often undergo incomplete cell divisions, resulting in multinucleated cells. We now present evidence that multinucleated glioma cells result from the functional loss of transient receptor potential canonical 1 (TRPC1) channels, plasma membrane proteins involved in agonist-induced calcium entry and reloading of intracellular Ca(2+) stores. Pharmacological inhibition or shRNA mediated suppression of TRPC1 causes loss of functional channels and store-operated calcium entry in D54MG glioma cells. This is associated with reduced cell proliferation and, frequently, with incomplete cell division. The resulting multinucleated cells are reminiscent of those found in patient biopsies. In a flank tumor model, tumor size was significantly decreased when TRPC1 expression was disrupted using a doxycycline inducible shRNA knockdown approach. These results suggest that TRPC1 channels play an important role in glioma cell division most likely by regulating calcium signaling during cytokinesis.

Fig. 1

TRPC inhibitors decrease glioma proliferation and store-operated calcium entry. A: Whole-cell patch-clamp recordings were used to isolate currents sensitive to TRPC inhibitors. The drug-sensitive currents were plotted as a function of applied voltage. Currents were linear and reversed at a potential of ~−25 mV. About 100 μM 2-APB (n = 6), 100 μM MRS1845 (n = 6), and 25 μM SKF96365 (n = 12) blocked identical currents. B: Proliferation assays (n = 5) revealed that channel inhibition with each of three inhibitors decreased glioma proliferation. By day 5, control cells (0.1% DMSO) grew to 42.24 ± 4.19-fold their starting cell number, whereas MRS1845 treatment reduced growth to 24.5 ± 6.5-fold. 2-APB and SKF treatments resulted in complete growth inhibition (0.54 ± 0.16-fold and 1.19 ± 0.24-fold, respectively). C: Immunocytochemical staining of D54MG cells using α-tubulin (green), TRPC1 (red), and DAPI (blue) staining and treated with either SKF or without drug for 2 days. SKF treatment resulted in larger cells with frequent multinucleation. Scale bars are 20 μm. D: Plots of Fura-2 imaging using the SOCE protocol (see Methods section). Two minutes before calcium addition, we applied either 0.1% DMSO (control) (n = 121) or the TRPC inhibitors 2-APB (n = 88), MRS1845 (n = 109), and SKF (n = 108). SOCE was significantly decreased (P < 0.05) with all TRPC inhibitors. The SOCE protocol was repeated with a TRPC1-blocking antibody applied for the entire imaging protocol (n = 108), and SOCE was reduced (P < 0.05) when compared with no drug controls (n = 100) or a nonblocking TRPC5 antibody (n = 75).

Fig. 2

Constitutive TRPC1 shRNA plasmids decrease TRPC1 protein expression, SKF-sensitive currents, and store-operated calcium entry. A: Representative Western blots of puromycin selected D54MG cells transfected with NS or TRPC1 shRNA plasmids showed that TRPC1 protein expression is decreased. Antibodies to TRPC5, Orai1, and Stim1 revealed no change in the protein level of other purported SOCE channel members. B: Quantification of TRPC1 protein knockdown (n = 4) revealed a 50–60% decrease in TRPC1 expression. C: Using whole-cell patch-clamp recordings, we isolated SKF-sensitive currents from TRPC1 shRNA cells transfected cells. The NS control plasmid (n = 12) displayed a prominent SKF-sensitive current while either two shRNA plasmids (n = 9 each), by contrast, did not display currents. D: Fura-2 imaging on transfected D54MG cells using SOCE protocol (see Methods section). Transient transfections were used and indicated by GFP expression. There was no difference in SOCE between control plasmid transfected (n = 71), control plasmid nontransfected (n = 109), shRNA1 nontransfected (n = 117), or shRNA2 nontransfected (n = 114). Cells transfected with TRPC1 shRNA (n = 45) or TRPC1 shRNA2 (n = 42) had a decrease (P < 0.05) in SOCE. Peak fluorescence data indicated SOCE decreased by 25–30% when TRPC1 shRNA was transfected.

Fig. 3

Inducible TRPC1 shRNA plasmids decrease TRPC1 protein expression and channel function. A: Representative Western blot displaying reduced TRPC1 protein expression in shRNA transfected cells when doxycycline treated for 5 days. B: Using whole-cell patch-clamp electro-physiology, SKF-sensitive currents from TRPC1 shRNA cells induced with doxycycline for 4–6 days (n = 19) were decreased (P < 0.05) when compared with control plasmid noninduced (n = 17), control plasmid induced (n = 16), or TRPC1 shRNA plasmid noninduced (n = 14). C: Mean electro-physiological data from the same cells revealed no change in paxilline-sensitivity (BK channels). D: Doxycycline treatment on TRPC1 shRNA transcription resulted in larger cells, measured by whole-cell capacitance (P < 0.05). Control plasmid noninduced were 44.33 ± 1.56 pF, control plasmid induced were 46.93 ± 1.61 pF, and shRNA plasmid noninduced were 39.2 ± 1.47 pF. The induced TRPC1 shRNA cells were 86.89 ± 3.7 pF.

Fig. 4

Doxycycline-induced TRPC1 shRNA impairs store-operated calcium entry and glioma proliferation. A: Fura-2 imaging on pTRIPZ transfected cells using SOCE protocol (see Methods section) showed no difference between control plasmid noninduced (n = 215), control plasmid induced (n = 234), or TRPC1 shRNA noninduced (n = 228). When TRPC1 shRNA cells were treated with doxycycline for 4–5 days (n = 255), SOCE was reduced (P < 0.05). B: Peak fluorescence data indicated a 24.1% decrease in SOCE when TRPC1 shRNA was induced with doxycycline. C: Proliferation assays (n = 6) with inducible control and shRNA plasmids were performed (see Methods section). After 6 days of treatment with or without doxycycline, cells proliferated 46.75 ± 4.28-fold (noninduced control), 40.77 ± 4.03-fold (induced control), and 39.24 ± 3.12-fold (noninduced TRPC1 shRNA) their starting cell number. Induced TRPC1 shRNA cells only proliferated to 7.26 ± 1.03-fold their starting cell number.

Fig. 5

Interference with TRPC1 channel function results in higher percentages of multinucleated cells. Representative stainings of D54MG cells with FITC-tubulin identified the mitotic spindle within a (A) noninduced control cell, induced control cell, or noninduced TRPC1 shRNA cell. FITC-tubulin staining of induced TRPC1 shRNA cells revealed a different morphology including multinucleated cells. Scale bars are 20 μm. B: There were fewer cells in mitosis (P < 0.05), with mitotic spindles, when TRPC1 shRNA was induced when compared with noninduced shRNA cells. There was no difference between control plasmid cells treated with or without doxycycline. C: Induced TRPC1 shRNA cells had significantly more (P < 0.05) multinucleated cells (13% ± 2%) when compared with control noninduced (1% ± 0.1%), control induced (1% ± 0.1%), or TRPC1 shRNA noninduced (1% ± 0.1%). D: We observed normal mitosis from TRPC1 shRNA cells without doxycycline where, in positions 1–5, they started flat, the cell rounded up into mitosis, the cell began to divide, and two daughter cells emerged (4 and 5). In induced TRPC1 shRNA cells, we observed instances of incomplete cell division where, in positions 1–5, they started flat, the cell rounded up into mitosis, the cell began to divide, and one cell emerged (4) and bizarre nuclei were observed (5). Scale bars are 20 μm.

Fig. 6

Decreased TRPC1 protein expression correlates with an increased percentage of multinucleated cells in GBM patient biopsies. Glioblastoma patient biopsy lysates were analyzed using western blot for TRPC1 expression in tissue from patients with a small (+), medium (++), or large (+++) percentage of large, multinucleated cells. As the number of large, multinucleated cells increased, TRPC1 channel expression decreased when compared with GAPDH-loading control.

Fig. 7

Decreased TRPC1 channel function impairs in vivo tumor growth. (A) Scatter and (B) line plot of flank tumor size of nude mice fed regular or doxycycline supplemented chow. After 31 days, the mice fed doxycycline to silence TRPC1 (n = 12) had significantly smaller tumors (P < 0.05) than their litter-mates fed regular food (n = 11). These tumors were still smaller (P < 0.05) at 35 days when the animals were sacrificed. C: Photographs show representative mice from noninduced and induced conditions of the largest and smallest tumors of each group. Some mice fed doxycycline had shallow tumors. D: The excised tumor from mice was measured again and a depth measurement obtained. Tumors from mice fed doxycycline were 37% smaller (P < 0.05).