Cysteine transporter SLC3A1 promotes breast cancer tumorigenesis

Abstract

Cysteine is an essential amino acid for infants, aged people as well as patients with metabolic disorders. Although the thiol group of cysteine side chain is active in oxidative reactions, the role of cysteine in cancer remains largely unknown. Here, we report that the expression level of the solute carrier family 3, member 1 (SLC3A1), the cysteine carrier, tightly correlated with clinical stages and patients' survival. Elevated SLC3A1 expression accelerated the cysteine uptake and the accumulation of reductive glutathione (GSH), leading to reduced reactive oxygen species (ROS). ROS increased the stability and activity of PP2Ac, resulting in decreased AKT activity. Hence, SLC3A1 activated the AKT signaling through inhibiting PP2A phosphatase activity. Consistently, overexpression of SLC3A1 enhanced tumorigenesis of breast cancer cells, whereas blocking SLC3A1 either with specific siRNA or SLC3A1 specific inhibitor sulfasalazine suppressed tumor growth and also abolished dietary NAC-promoted tumor growth. Collectively, our data demonstrate that SLC3A1 promotes cysteine uptake and determines cellular response to antioxidant N-acetylcysteine, suggesting SLC3A1 is a potential therapeutic target for breast cancer.

Keywords: PDK1; ROS; breast cancer; cysteine; solute carrier SLC3A1.

Figure 1

The expression level of SLC3A1 is correlated with breast cancer progression. A. Expression analysis of cysteine transporter genes by qPCR in breast cancer cell lines. B. Immunohistochemistry analysis on tissue array of clinical breast cancers vs peritumoral tissues. The graphic presents the levels of SLC3A1 expression, which were analyzed by the densitometry method (***: P < 0.001). C. Immunohistochemistry analysis on tissue array of breast cancers at different clinical stages. The graphic presents the levels of SLC3A1 expression, analyzed by the densitometry method (***: P < 0.001). D. Kaplan-Meier analysis of breast cancer patients with low level (259 cases) vs high level (132 cases) of SLC3A1 expression.

Figure 2

SLC3A1 promotes tumorigenesis of breast cancer cells. A. SLC3A1 expression analysis of breast cancer cell lines. B. SLC3A1 overexpression increases MDA-MB-231 cell growth (*: P < 0.05); SLC3A1 knockdown decreases MDA-MB-453 cell growth (*: P < 0.05). The left pictures were representative cell photos at the indicated time points, the right histograms were quantitative results. C. SLC3A1 promotes the colony formation of breast cancer cells (**: P < 0.01; *: P <0.05). D. SLC3A1 overexpression promotes breast cancer cell tumorigenesis in xenograft mouse model. Blue is the control, Red is the overexpression group. E. SLC3A1 knockdown attenuates MDA-MB-453 cells tumorigenesis. Blue is control.

Figure 3

SLC3A1 decreased ROS level by elevating cysteine uptake. A. SLC3A1 increases Cysteine uptake. The cellular concentration of amino acid except cysteine was analyzed by GC-MS. Cysteine uptake was analyzed using the Homocysteine ELISA Kit. The cysteine concentration in control cells was 17.56 mg/L. B. SLC3A1 increases the content of reductive GSH and the ratio of GSH to GSSH. C. SLC3A1 increases the cellular ROS level. The Sulfasalazine was used a SLC3A1 inhibitor. NAC: N-acetylcysteine.

Figure 4

SLC3A1 promoted AKT activation by suppressing PP2A activity. A. Screening for potential molecule(s) mediating SLC3A1 regulation of tumor growth and progression. B. AKT activation induced by SLC3A1. The plasmid expressing SLC3A1 were transfected into 293T cells at the indicated concentrations. C. SLC3A1-triggered AKT activation abolished by H₂ O₂. D. PP2Ac but not PDK1 was regulated by cellular ROS level. E. PP2Ac was increased in breast cancer MDA-MB-453 cells depleted of SLC3A1. F. Inhibition of PP2A enhances AKT activation. The final concentration of okadaic acid was 1nM. G. SLC3A1 promotes oncogene expression through AKT-β-catenin pathway. H. ROS increases PP2Ac stability and activity through cysteine oxidation. The PP2Ac proteins for activity assay were purified from 293T cells treated with or without H₂ O₂. The yellow marked residues are conserved substrate binding motif of protein tyrosine phosphatases. The red marked residues are a speculated ROS reaction motif. CHX (cycloheximide): a protein synthesis inhibitor.

Figure 5

Inhibition of SLC3A1 suppresses breast cancer tumorigenesis. A. Determination of dose and treating time of sulfasalazine (SASP) on breast cancer cell. MDA-MB-231 cells were treated for 4 days at the indicated doses in the dose response experiment or treated with 1 mM of SASP for the indicated time points. B. Pre-treatment with SLC3A1 inhibitor attenuated tumor initiation and tumor growth of breast cancer. Tumors in blue circles but not in red circles were inoculated with MDA- MB-231 cells pre-treated with SLC3A1 inhibitor sulfasalazine. C. SLC3A1 inhibitor sulfasalazine suppressed tumor growth. SASP: Sulfasalazine, 16 mg/Kg/Day for 10 days. NAC: N-acetylcysteine, 1g/L in drinker water. D. The working model for SLC3A1 determination of cysteine promotion of breast cancer tumorigenesis. Cys: cysteine; ROS: reactive oxidative species; PP2A: protein serine/threonne phosphatase 2A.