The plasma membrane transporter SLC5A8 suppresses tumour progression through depletion of survivin without involving its transport function

Abstract

SLC5A8 (solute carrier gene family 5A, member 8) is a sodium-coupled transporter for monocarboxylates. Among its substrates are the HDAC (histone deacetylase) inhibitors butyrate, propionate and pyruvate. Expression of SLC5A8 is silenced in cancers via DNA methylation, and ectopic expression of SLC5A8 in cancer cells induces apoptosis in the presence of its substrates that are HDAC inhibitors. In the present study we show that ectopic expression of SLC5A8 in cancer cells translocates the anti-apoptotic protein survivin to the plasma membrane through protein-protein interaction resulting in depletion of nuclear survivin and also decreases cellular levels of survivin through inhibition of transcription. These SLC5A8-induced changes in the location and levels of survivin result in cell-cycle arrest, disruption of the chromosome passenger complex involved in mitosis, induction of apoptosis and enhancement in chemosensitivity. These effects are seen independently of the transport function of SLC5A8 and histone acetylation status of the cell; in the presence of pyruvate, a SLC5A8 substrate and also an HDAC inhibitor, these effects are amplified. Ectopic expression of SLC5A8 in the breast cancer cell line MB231 inhibits the ability of cells to form colonies in vitro and to form tumours in mouse xenografts in vivo. The suppression of survivin transcription occurs independently of HDAC inhibition, and the underlying mechanism is associated with decreased phosphorylation of STAT3 (signal transducer and activator of transcription 3). The observed effects are specific for survivin with no apparent changes in expression of other inhibitor-of-apoptosis proteins. The present study unravels a novel, hitherto unrecognized, mechanism for the tumour-suppressive role of a plasma membrane transporter independent of its transport function.

Figure 1

SLC5A8 decreases survivin mRNA and protein. A, Semi-quantitative RT-PCR of relative mRNA levels of survivin and SLC5A8 in MCF10A series cell lines: MCF10A-I (normal epithelium), MCF10A-II (oncogenically initiated premalignant epithelium), MCF10A-III (low grade carcinoma), and MCF10A-IV (high-grade metastatic carcinoma). GAPDH was used as internal control. B, MCF10A-IV cells were made to stably express SLC5A8 through lentiviral transfection. RNA was isolated and expression of survivin was analyzed by RT-PCR. C and D, The expression of SLC5A8 was induced with doxycycline in MCF7-SLC5A8-TetOn and MB231-SLC5A8-TetOn for 72 h. Pyruvate (1 mM) was added to the medium after 48 h of doxycycline treatment as indicated. At 72 h, expression of survivin was analyzed by RT-PCR (C) and immunoblotting (D). β-Actin was used as an internal control for Western blot.

Figure 2

SLC5A8 decreases survivin levels in nucleus through sequestration to plasma membrane. A, Cells were treated with 3 µg/ml of 5’-azacytidine and expression of SLC5A8 and survivin was analyzed by semi-quantitative RT-PCR. B, The expression of SLC5A8 was induced with doxycycline in MB231-SLC5A8-TetOn for 3 days. After 3 days, doxycycline was removed and the cells were cultured for an additional 3 days. RNA was isolated at day 3 and 6, and survivin and SLC5A8 expression was analyzed by semi-quantitative RT-PCR. C, Survivin was immunoprecipitated from MB231-SLC5A8 cells using anti-survivin antibody and immunoblotted with anti-SLC5A8 antibody. D, The expression of SLC5A8 was induced with doxycycline in MCF7-SLC5A8-TetOn cells for 72 h. Corresponding vector-only MCF7 cells were also used in parallel. Pyruvate (1 mM) was added to the indicated cells after 48 h of doxycycline treatment. Cells were fixed at 72 h and immunofluorescence analysis was carried out with anti-survivin and anti-SLC5A8 antibodies and visualized with Alexafluor 488- and Alexafluor 568-conjugated secondary antibodies respectively. Hoechst was used to locate the nucleus. Scale bar represents 10 µm.

Figure 3

Depletion of survivin by SLC5A8 impairs CPC formation. A, MB231-SLC5A8 cells were synchronized in G1 phase with double thymidine block and released in fresh media with 10% FBS. The progression through cell cycle was followed at indicated time points using propidium iodide staining. Values presented are means of three independent experiments. B, MB231-SLC5A8 cells were synchronized in mitotic phase with nocodazole, and survivin was immunoprecipitated using anti-survivin antibody and immunoblotted with anti-Aurora B antibody. C, MB231-SLC5A8 cells were synchronized in mitotic phase with nocodazole, and immunofluorescence analysis was carried out with specified antibodies. Hoechst was used to locate the nucleus. Scale bar represents 5µm. D, Binucleated cells were analyzed using Cell mask stain in MB231-SLC5A8 and MCF7-SLC5A8 and corresponding vector cells. Scale bar represents 10µm. Binucleated cells were counted in six independent fields and are depicted in (E). Data are presented as means ± SE. a, p<0.05.

Figure 4

SLC5A8 increases cell death in breast cancer cells and decreases tumor formation in mouse xenografts. A, Caspase 3 activity was increased in MB231-SLC5A8 cells compared to control vector-only cells. Values (means ± SE) are from three independent experiments. a, p<0.05. B, Immunoblot analysis of cleaved PARP and active caspase 3 in MB231-SLC5A8 and vector-only cells. C, Flowcytometric analysis of Annexin V-propidium iodide positive cells in MB231-SLC5A8. Values (means ± SE) are from three independent experiments. a, p<0.05. D, MB231-SLC5A8 and vector-only cells (1×10⁷ cells) were injected into mammary pads of athymic nude mice, and the growth of tumor was monitored using a caliper. Inset shows the size of the tumors at the end of experimental period (left, vector-only MB231 cells; right, MB231-SLC5A8 cells). E, Colony formation assay was performed with vector-only MB231 cells and MB231-SLC5A8 cells in the presence and absence of pyruvate. The colonies were stained with Giemsa and quantified (F). a, p<0.05.

Figure 5

SLC5A8 increases chemosensitivity in breast cancer cells. MCF7-SLC5A8 and MB231-SLC5A8 cells and corresponding vector-only cells were treated with indicated concentrations of 5-fluorouracil or cisplatin for 72 h. The apoptotic and necrotic cells were quantified by Annexin Vpropidium iodide staining. Values (means ± SE) are from three independent experiments. *, p<0.05.

Figure 6

SLC5A8 expression has no effect on histone acetylation but decreases STAT3 phosphorylation in breast cancer cells. A, Immunoblot analysis of H4K16 and total H4 in MCF7-SLC5A8 and MB231-SLC5A8 cells and corresponding vector-only cells B, MB231-SLC5A8 cells and corresponding vectoronly cells were transfected with survivin-promoter and β-actin renilla luciferase constructs. After 48 h, the reporter activity was measured using the dual luciferase assay; data represent values after normalization with renilla luciferase. a, p<0.05. C, Immunoblot analysis of phosphorylated and total STAT3 in MCF7-SLC5A8 and MB231-SLC5A8 cells and corresponding vector-only cells. D, Immunofluorescence analysis of phosphorylated STAT3 in MB231-SLC5A8 cells and corresponding vector-only cells. Scale bar represents 10 µm.