TTYH1 and TTYH2 Serve as LRRC8A-Independent Volume-Regulated Anion Channels in Cancer Cells

Abstract

Volume-regulated anion channels (VRACs) are involved in cellular functions such as regulation of cell volume, proliferation, migration, and cell death. Although leucine-rich repeat-containing 8A (LRRC8A) has been characterized as a molecular component of VRACs, here we show that Drosophila melanogaster tweety homologue 1 and 2 (TTYH1 and TTYH2) are critical for VRAC currents in cancer cells. LRRC8A-independent VRAC currents were present in the gastric cancer cell line SNU-601, but almost completely absent in its cisplatin-resistant derivative SNU-601-R10 (R10). The VRAC current in R10 was partially restored by treatment with trichostatin A (TSA), a histone deacetylase inhibitor. Based on microarray expression profiling of these cells, we selected two chloride channels, TTYH1 and TTYH2, as VRAC candidates. VRAC currents were completely absent from TTYH1- and TTYH2-deficient SNU-601 cells, and were clearly restored by expression of TTYH1 or TTYH2. In addition, we examined the expression of TTYH1 or TTYH2 in several cancer cell lines and found that VRAC currents of these cells were abolished by gene silencing of TTYH1 or TTYH2. Taken together, our data clearly show that TTYH1 and TTYH2 can act as LRRC8A-independent VRACs, suggesting novel therapeutic approaches for VRACs in cancer cells.

Keywords: LRRC8A; TTYH1; TTYH2; VRAC; cancer cells.

Figure 1

Volume-activated chloride currents in SNU-601 cells. (a) Representative traces showing time courses of the volume-activated chloride current in SNU-601 and R10 cells elicited by voltage ramp from −100 to +100 mV. (b) Representative traces showing the current–voltage relationship for volume-activated chloride currents in SNU-601 and R10 cells before and during perfusion with hypotonic solution, respectively. (c) Summary bar graph showing the ratio of current amplitudes of SNU-601 (n = 7) and R10 cells (n = 7) before and during perfusion with a hypotonic solution. (d) Representative traces of volume-regulated anion channel (VRAC) currents of SNU-601 cells before and during perfusion with a hypotonic solution, and during DCPIB application in a hypotonic solution. (e) Summary bar graph showing the ratio of current amplitudes of DCPIB-sensitive currents before and after DCPIB application (n = 7). Data are presented as means ± SEM (*** P < 0.001).

Figure 2

SNU-601 cells have a LRRC8A-independent VRAC activity. (a) Representative traces showing the current–voltage relationship for VRACs in SNU-601 cells transfected with scrambled or LRRC8A shRNAs under isotonic or hypotonic conditions. (b) Summary bar graph showing the ratio of current amplitudes of hypotonic/isotonic solutions in SNU-601 cells transfected with scrambled or LRRC8A shRNAs (n = 6). (c) Representative traces showing the current–voltage relationship for VRACs in HEK293T cells transfected with scrambled or LRRC8A shRNAs under isotonic or hypotonic conditions. (d) Summary bar graph showing the ratio of current amplitudes of hypotonic/isotonic solutions in SNU-601 cells transfected with scrambled shRNA (n = 5) or LRRC8A shRNA (n = 11). (e) Real-time PCR quantification of fold changes in LRRC8 family mRNAs in SNU-601 and R10 cells. The experiments were repeated three times. Data are presented as means ± SEM (** P < 0.01, *** P < 0.001, n.s, not significant).

Figure 3

TTYH1 and TTYH2 are candidate VRACs in SNU-601 cells. (a) Representative traces showing the time course of volume-activated chloride currents elicited by alternating pulses by voltage ramp from −100 to +100 mV, in R10 and TSA-treated R10 cells. (b) Schematic overview of our human whole-genome array screen to identify candidate VRAC components. (c) Scatter plots of log2 (Raw) for every gene in SNU-601 vs. R10 and TSA-treated R10 vs. R10. (d) Dot plots of expression levels of TTYH1, TTYH2, and CFTR in SNU-601, R10, and TSA-treated R10 cells samples, calculated rom array data. (e) Real-time PCR quantification of fold changes in TTYH1, TTYH2, and CFTR mRNAs in SNU-601, R10, and TSA-treated R10 cells. Data are presented as means ± SEM (** P < 0.01).

Figure 4

VRAC activity is completely abolished in dKO cells, and is restored by expression of TTYH1 or TTYH2. (a) Western blot data for TTYH1 and LRRC8A (SWELL1) in SNU-601 and TTYH1 and TTYH2 double-knockout (dKO) cells. (b) Representative immunocytochemical images of SNU-601 and dKO cells stained with anti-TTYH2 antibody. (c) Representative traces of VRAC currents from dKO cells transfected with GFP controls in isotonic and hypotonic solutions. Notably, VRAC currents were not induced by hypotonic stimulation in these cells. (d) Representative traces of VRAC currents from dKO cells transfected with TTYH1-GFP in isotonic and hypotonic solutions. (e) Representative traces of VRAC currents from dKO cells transfected with TTYH2-GFP in isotonic and hypotonic solutions. (f) Representative traces of VRAC currents from dKO cells co-transfected with TTYH1-GFP and TTYH2-GFP in isotonic and hypotonic solutions. (g) Summary bar graph showing current densities in isotonic and hypotonic solutions, as in (c)–(f), at +100 mV. Data are presented as means ± SEM (** P < 0.01 and *** P < 0.001).

Figure 5

VRAC currents are observed in cancer cell lines expressing TTYH1 or TTYH2. (a) Expression of TTYH1 and TTYH2 mRNA in various cancer cell lines was measured by RT-PCR. (b) Representative traces of VRAC currents from HepG2 cells in isotonic and hypotonic solutions. (c) Summary bar graph showing current densities of HepG2 cells in isotonic and hypotonic solutions, as in (b), at +100 mV. (d) Representative traces of VRAC currents from LoVo cells in isotonic and hypotonic solutions. (e) Summary bar graph showing current densities of LoVo cells in isotonic and hypotonic solutions, as in (d), at +100 mV. (f) Representative traces of VRAC currents from MCF-7 cells in isotonic and hypotonic solutions. (g) Summary bar graph showing current densities of MCF-7 cells in isotonic and hypotonic solutions, as in (f), at +100 mV. Data are presented as means ± SEM (* P < 0.05, ** P < 0.01, **** P < 0.0001).

Figure 6

Lack of TTYH1 or TTYH2 alone in HepG2 or LoVo cells almost completely eliminates VRAC activity. (a) Efficiency of gene silencing by the TTYH1 shRNA construct was monitored by RT-PCR in HepG2 cells. (b) Representative traces of VRAC currents from HepG2 cells transfected either with scrambled or TTYH1 shRNA in isotonic and hypotonic solutions. (c) Summary bar graph showing current densities of transfected HepG2 cells in isotonic and hypotonic solutions, as in (b), at +100 mV. (d) Efficiency of gene silencing by the TTYH2 shRNA construct was monitored by RT-PCR. (e) Representative traces of VRAC currents from LoVo cells transfected with scrambled or TTYH2 shRNA in isotonic and hypotonic solutions. (f) Summary bar graph showing current densities of transfected LoVo cells in isotonic and hypotonic solutions, as in (e), at +100 mV. Data are presented as means ± SEM (** P < 0.01, **** P < 0.0001).