Prostate Cancer Cells in Different Androgen Receptor Status Employ Different Leucine Transporters

BACKGROUND: Leucine stimulates cancer cell proliferation through the mTOR pathway, therefore, inhibiting leucine transporters may be a novel therapeutic target for cancer. L-type amino acid transporter (LAT) 1, a Na⁺-independent amino acid transporter, is highly expressed in many tumor cells. However, leucine transporter(s) in different stages of prostate cancer, particularly in the stages of castration resistance with androgen receptor (AR) expression, is unclear. METHODS: LNCaP and DU145 and PC-3 cell lines were used as a model of androgen dependent, and metastatic prostate cancer. A new “LN-cr” cell line was established after culturing LNCaP cells for 6 months under androgen-free conditions, which is considered a model of castration resistant prostate cancer (CRPC) with androgen AR expression. The expression of leucine transporters was investigated with quantitative PCR and immunofluorescence. Uptake of ¹⁴C Leucine was examined in the presence or absence of BCH (a pan-LAT inhibitor), JPH203 (an LAT1-specific inhibitor), or Na⁺. Cell growth was assessed with MTT assay. siRNA studies were performed to evaluate the indispensability of y⁺LAT2 on leucine uptake and cell viability in LN-cr. RESULTS: Cell viability showed a 90% decrease in the absence of leucine in all four cell lines. LNCaP cells principally expressed LAT3, and their leucine uptake was more than 90% Na⁺-independent. BCH, but not JPH203, inhibited leucine uptake, and cell proliferation (IC_50BCH:15 mM). DU145 and PC-3 cells predominantly expressed LAT1. Leucine uptake and cell growth were suppressed by BCH or JPH203 in a dose-dependent manner (IC_50BCH: ∼20 mM, IC_50JPH203: ∼5 µM). In LN-cr cells, Na⁺-dependent uptake of leucine was 3.8 pmol/mgprotein/min, while, Na⁺-independent uptake was only 0.52 (P < 0.05). Leucine uptake of LN-cr was largely (∼85%) Na⁺-dependent. y⁺LAT2 expression was confirmed in LN-cr. Knockdown of y⁺LAT2 lead to significant leucine uptake inhibition (40%) and cell growth inhibition (20%). CONCLUSIONS: New CRPC cell line with increased expression of y⁺LAT2 as a leucine transporter was established in vitro. Anti-leucine transporter therapy could be an important option against prostate cancer. Prostate 77:222–233, 2017.