SLC9A3R1 stimulates autophagy via BECN1 stabilization in breast cancer cells

Abstract

Autophagy, a self-catabolic process, has been found to be involved in abrogating the proliferation and metastasis of breast cancer. SLC9A3R1 (solute carrier family 9, subfamily A [NHE3, cation proton antiporter 3], member 3 regulator 1), a multifunctional scaffold protein, is involved in suppressing breast cancer cells proliferation and the SLC9A3R1-related signaling pathway regulates the activation of autophagy processes. However, the precise regulatory mechanism and signaling pathway of SLC9A3R1 in the regulation of autophagy processes in breast cancer cells remains unknown. Here, we report that the stability of BECN1, the major component of the autophagic core lipid kinase complex, is augmented in SLC9A3R1-overexpressing breast cancer MDA-MB-231 cells, subsequently stimulating autophagy by attenuating the interaction between BECN1 and BCL2. Initially, we found that SLC9A3R1 partially stimulated autophagy through the PTEN-PI3K-AKT1 signaling cascade in MDA-MB-231 cells. SLC9A3R1 then attenuated the interaction between BECN1 and BCL2 to stimulate the autophagic core lipid kinase complex. Further findings revealed that SLC9A3R1 bound to BECN1 and subsequently blocked ubiquitin-dependent BECN1 degradation. And the deletion of the C-terminal domain of SLC9A3R1 resulted in significantly reduced binding to BECN1. Moreover, the lack of C-terminal of SLC9A3R1 neither reduced the ubiquitination of BECN1 nor induced autophagy in breast cancer cells. The decrease in BECN1 degradation induced by SLC9A3R1 resulted in the activity of autophagy stimulation in breast cancer cells. These findings indicate that the SLC9A3R1-BECN1 signaling pathway participates in the activation of autophagy processes in breast cancer cells.

Keywords: SLC9A3R1 signaling pathway, stimulating autophagy; breast cancer cells; protein interaction; protein stability; ubiquitination degradation.

Figure 1.

Figure 1 (See previous page). SLC9A3R1 stimulates autophagy activity in MDA-MB 231 cells. (A) Overexpression of SLC9A3R1 upregulated the expression of autophagy-related proteins. MDA-MB-231 cells stably expressing vector or MYC-SLC9A3R1 were harvested and autophagy-related proteins were detected by western blot analysis. ACTB served as the loading control. The value for the vector was set to 1.0 and the other values were normalized. (B) Overexpression of SLC9A3R1 aggregated LC3 foci. MDA-MB-231 cells stably expressing vector or MYC-SLC9A3R1 were fixed in 4% paraformaldehyde, stained with fluorochromes, and imaged by confocal microscopy, Scale bar: 20 μm. (C) Statistical analysis of the numbers of LC3 dots per cell in (B). Quantification of LC3 dots per cell was done as described in the Materials and Methods. (D) Electron microscopy of MDA-MB-231 cells stably expressing vector or MYC-SLC9A3R1. Typical autophagosome observed in SLC9A3R1-overexpressing breast cancer cells. The lysosome is indicated by the arrow, and the autophagosome is indicated by the arrowhead. Magnification × 12, 000–30, 000. (E) Statistical analysis of the numbers of autophagosomes per 100 μm² in (D). (F) Treatment with CQ augmented the expression of LC3B-II. MDA-MB-231 cells expressing either vector or MYC-SLC9A3R1 were treated with or without CQ (50 μM) for 12 h, and the expression of LC3B-II was measured by western blotting. Data are presented as the mean ± SE of 4 independent assays. *, P < 0.05; **, P < 0.01; ***, P < 0.001; N, nucleus; APs, autophagosomes; CQ, chloroquine.

Figure 2

(See previous page). SLC9A3R1 partially stimulates autophagy through the PTEN-PI3K-AKT1 pathway. (A–D) SLC9A3R1 is mostly distributed in the cytoplasm of breast cancer cells. For panels (A and C), breast cancer cells were harvested, and the subcellular fraction was extracted for immunoblotting. ACTB and LMNA were used as loading controls. For panels (B and D), breast cancer cells in dishes were fixed in 4% paraformaldehyde, stained with fluorochromes, and imaged by confocal microscopy, Scale bar: 20 μm. For panels (A and B), the experiments were carried out in MDA-MB-231 cells stably expressing vector or MYC-SLC9A3R1. For panels (C and D), the experiments were carried out in MCF-7 cells stably expressing control-shRNA or sh-SLC9A3R1. (E) SLC9A3R1 stimulates the PTEN-PI3K-AKT1 pathway in MDA-MB-231 cells. MDA-MB-231 cells stably expressing vector or MYC-SLC9A3R1 were harvested, and the expression of the indicated proteins was determined by immunoblotting. The value for the vector was set to 1.0 and the other values were normalized. (F) Silencing of SLC9A3R1 suppresses the PTEN-PI3K-AKT1 pathway in MCF-7 cells. MCF-7 cells stably expressing control-shRNA or sh-SLC9A3R1 were harvested, and the expression of the indicated proteins was detected by immunoblotting. The value for the control-shRNA was set to 1.0 and the other values were normalized. (G) PTEN is unnecessary for the activation of autophagy when SLC9A3R1 stimulates. The MDA-MB-231 cells were transfected with control-siRNA or si-PTEN. Cell lysates were collected, and the expression of autophagy-related proteins was analyzed by western blotting. The value for the vector was set to 1.0 and the other values were normalized. Data are presented as the mean ± SE (n = 4). *, P < 0.05; ***, P < 0.001.

Figure 3.

SLC9A3R1 activates the autophagic core lipid kinase complex by inducing the expression of BECN1. (A) SLC9A3R1 increases the expression of BECN1 in MDA-MB-231 cells. MDA-MB-231 cells were transfected with the indicated concentrations of MYC-SLC9A3R1 or vector and protein lysates were analyzed by immunoblotting with the indicated antibodies. (B) Knockdown of SLC9A3R1 decreases the expression of BECN1 in MCF-7 cells. MCF-7 cells were transfected with the indicated concentrations of si-SLC9A3R1 or control-siRNA, and protein lysates were analyzed by immunoblotting with the indicated antibodies. (C, D) SLC9A3R1 reduces the binding between BECN1 and BCL2. MDA-MB-231 cells were transfected with HA-BECN1, Flag-BCL2, and MYC-SLC9A3R1 plasmids for 24 h; cell lysates were then prepared and immunoprecipitated with anti-HA antibody or equal amount of mouse IgG, and the precipitates were detected using an anti-Flag antibody. Data are presented as the mean ± SE (n = 4). ***, P < 0.001.

Figure 4.

SLC9A3R1 inhibits the degradation of BECN1. (A, B) SLC9A3R1 did not affect BECN1 mRNA in breast cancer cells. Cells were transfected with the indicated concentrations of MYC-SLC9A3R1 (A) or si-SLC9A3R1 (B), and total RNA was isolated. The BECN1 mRNA was analyzed by fluorescent quantitative RT-PCR, as indicated in Materials and Methods. (C) SLC9A3R1 inhibits the degradation of BECN1 in MDA-MB-231 cells. Cells were transfected with HA-BECN1 for 24 h, subsequently the cells were then treated with CHX (20 μmol/L) for the indicated times. The cell lysates were then examined by western blotting using an anti-HA antibody. Cells were also transfected with an equal amount of pEGFP-N1 plasmid to monitor the transfection efficiency. (D) Knockdown of SLC9A3R1 promotes the degradation of BECN1 in MCF-7 cells. MCF-7 cells overexpressing HA-tagged BECN1 by lentivirus vector were treated with CHX (20 μmol/L) for the indicated times. The cell lysates were then analyzed by western blotting using anti-HA antibodies. Data are presented as the mean ± SE (n = 4). NS, nonsignificant; CHX, cycloheximide; GFP, green fluorescent protein.

Figure 5.

SLC9A3R1 inhibits the proteasomal degradation of BECN1. (A) MG132 inhibition of the proteasome blocks the degradation of BECN1 induced by SLC9A3R1 depletion. MCF-7 cells were overexpressed HA-tagged BECN1 by lentivirus vector and treated with MG132 (10 μmol/L) for 2 h and then treated with CHX (20 μmol/L) for the indicated times. (B) CQ inhibition of the lysosome does not affect the degradation of BECN1 induced by SLC9A3R1 depletion. MCF-7 cells were overexpressed HA-tagged BECN1 by lentivirus vector and treated with CQ (100 μmol/L) for 12 h and then treated with CHX (20 μmol/L) for the indicated times. (C) Overexpression of SLC9A3R1 eliminates the ubiquitination of BECN1. MDA-MB-231 cells were transfected with Flag-ubiquitin, HA-BECN1 and MYC-SLC9A3R1 expression plasmids for 24 h and then treated with MG132 (10 μmol/L) for 2 h; the cell lysates were immunoprecipitated using an anti-HA antibody, and the precipitates were probed with anti-ubiquitin and anti-HA antibodies. CHX, cycloheximide; CQ, chloroquine; UB, ubiquitin.

Figure 6.

SLC9A3R1 inhibits the ubiquitin-dependent degradation of BECN1 by binding to BECN1. (A) SLC9A3R1 interacts with BECN1. MYC-SLC9A3R1 was cotransfected with HA-BECN1 into MDA-MB-231 cells. Whole-cell lysates were immunoprecipitated with an anti-HA antibody and blotted with an anti-MYC antibody. (B) In vitro binding between SLC9A3R1 and BECN1. The recombinant SLC9A3R1 full-length protein was incubated with an equal amount of GST or GST-BECN1 and analyzed by western blotting using an anti-SLC9A3R1 antibody. The presence of the GST fusion proteins was confirmed by western blotting using an anti-GST antibody. (C) Deficiency of the BCL2-binding domain of BECN1 inhibits the interaction between SLC9A3R1 and BECN1. Top: deletion mutants of BECN1. Below: MDA-MB-231 cells were cotransfected with MYC-SLC9A3R1 and the indicated constructs of HA-BECN1. Whole-cell extracts were immunoprecipitated with an anti-HA antibody. (D) SLC9A3R1 does not affect the ubiquitination of BECN1 lacking a BCL2-binding domain. MDA-MB-231 cells were cotransfected with Flag-ubiquitin, MYC-SLC9A3R1 and the indicated constructs of HA-BECN1 and then treated with MG132 (10 μmol/L) for 2 h. The cell lysates were extracted and immunoprecipitated using an anti-HA antibody. The precipitates were then examined with anti-ubiquitin and anti-HA antibodies. UB, ubiquitin.

Figure 7.

SLC9A3R1 stimulates autophagy by binding and stabilizing BECN1 in breast cancer cells. (A) Deficiency of the C-terminal domain of SLC9A3R1 inhibits the interaction between SLC9A3R1 and BECN1. Top: deletion mutants of SLC9A3R1. Below: MDA-MB-231 cells were cotransfected with HA-BECN1 and the indicated constructs of MYC-SLC9A3R1. Whole-cell extracts were immunoprecipitated with an anti-HA antibody. (B) Deficiency of the C-terminal domain of SLC9A3R1 does not affect the ubiquitination of BECN1. MDA-MB-231 cells were cotransfected with Flag-ubiquitin, HA-BECN1 and the indicated constructs of MYC-SLC9A3R1. The cells were then treated with MG132 (10 μmol/L) for 2 h. The cell lysates were extracted and immunoprecipitated using an anti-HA antibody. The precipitates were then analyzed with anti-ubiquitin and anti-HA antibodies. (C) Deficiency of the C-terminal domain of SLC9A3R1 does not induce autophagy. MDA-MB-231 cells stably expressing MYC-SLC9A3R1 or the indicated constructs of MYC-SLC9A3R1 were harvested and autophagy-related proteins were detected by western blot analysis. (D) Schematic diagram of the mechanism of SLC9A3R1-mediated autophagy stimulation in breast cancer cells. SLC9A3R1 interacts with BECN1 and inhibits the ubiquitin degradation of BECN1. Thus, enhanced BECN1 expression stimulates the autophagic core lipid kinase complex in breast cancer cells. Data are presented as the mean ± SE (n = 4). **, P < 0.01; ***, P < 0.001. UB, ubiquitin.