Blockade of the amino acid transporter SLC6A14 suppresses tumor growth in colorectal Cancer

Abstract

Background: The amino acid transporter SLC6A14, which transports 18 of the 20 proteinogenic amino acids, is too low to be detected in healthy normal tissues but is significantly increased in some solid cancers. However, little is known about the roles of SLC6A14 in colorectal cancer (CRC).

Methods: The mRNA and protein levels of SLC6A14 were detected using TCGA database, real-time polymerase chain reaction, western blot, and tissue microarrays, respectively. Amino acids concentration was determined by LC-MS/MS. Cell proliferation and apoptosis were determined using MTT assay and flow cytometry. Transwell invasion assay and wound healing assay were employed to analyze cell migration and invasion. The protein levels of Akt-mTOR signaling pathway and MMPs proteins were detected by western blot.

Results: Both of the mRNA and protein levels of SLC6A14 were upregulated in CRC tissues, and the protein levels of SLC6A14 were closely related to the tumor cells differentiation: the higher the expression of SLC6A14 was, the poorer the differentiation of the tumor cells was. Further knockdown SLC6A14 with siRNA or treatment with α-MT in CRC cell lines reduced cell proliferation and migration in vitro and inhibited xenograft tumor growth in vivo. Mechanistically, SLC6A14 was demonstrated to regulate the expression and phosphorylation of Akt-mTOR, which mediates the promoting tumor growth function of SLC6A14. Blockade of SLC6A14 with α-MT inhibited the activation of mTOR signaling.

Conclusion: SLC6A14 was upregulated in CRC and could promote tumor progression by activating the Akt-mTOR signaling pathway, which may serve as an effective molecular target for the treatment of CRC.

Keywords: Amino acid transporter; Colorectal cancer; SLC6A14; mTOR; α-Methyltryptophan.

Fig. 1

Amino acid transporter expression analysis showed that SLC6A14 is upregulated in colorectal cancer. A The publicly available TCGA database was used to analyze the mRNA expression levels of SLC6A14, SLC3, and SLC7 family transporters in colorectal cancer. B mRNA expression of SLC6A14, SLC3, and SLC7 family transporters in 28 matched CRC cancer tissues and paired adjacent colon tissues was detected by RT-PCR, and GAPDH was used as the internal control. The P value was calculated by Student’s test. C Representative images of SLC6A14 protein expression in paired normal and colorectal cancer tissues by Western blots. D The relative protein levels of SLC6A14 in the paired normal and colorectal cancer tissues were analyzed, and β-actin was used as the internal control. E The expression of SLC6A14 was evaluated by Western blots in five CRC cell lines (HT29, Caco2, HCT116, SW620 and SW480), and the liver cancer cell line Huh7. The data are expressed as the mean ± SD of three independent experiments. *P < 0.05

Fig. 2

SLC6A14 was frequently upregulated in CRC and predicted poor differentiation. A Representative images of the protein expression of SLC6A14 generated from the immunohistochemistry-based tissue microarray containing 482 samples. B The protein expression of SLC6A14 was analyzed by H-score in 482 tissue samples: 94 normal mucosa, 65 adenoma, 302 colon cancer, and 21 liver cancer samples. C Representative images of the protein expression of SLC6A14 in different differentiation of CRC. D The H-score of SLC6A14 staining in well-differentiated, moderately differentiated, and poorly differentiated CRC. Comparisons among three or more groups were conducted using one-way ANOVA, *P < 0.05

Fig. 3

Knockdown or blockade of SLC6A14 inhibits cell growth. A Decreased protein expression of SLC6A14 in the HCT116 and Caco2 cells treated with siRNA was detected by western blots. B The relative protein levels of SLC6A14 in the HCT116 and Caco2 cells treated with siRNA were analyzed, and β-actin was used as the internal control. C The growth curve of the HCT116 and Caco2 cells that were transfected with SLC6A14-siRNA or blockade of SLC6A14 with α-MT, as determined by MTT assay. The data are expressed as the mean ± SD of three independent experiments. *P < 0.05

Fig. 4

Knockdown or blockade of SLC6A14 suppresses cell proliferation and promotes apoptosis. A Cell cycle analysis G₁/S transition of the HCT116 or Caco2 cells transfected with SLC6A14-siRNA or treated with α-MT by flow cytometry. B Quantification of cell cycle analysis. C The cell apoptosis of the HCT116 or Caco2 cells transfected with SLC6A14-siRNA or treated with α-MT cultured in FBS-free medium was stained with Annexin-V/PI and examined by flow cytometry. D The quantification of apoptotic cells identified as PI-negative and Annexin-V-positive staining. The data are expressed as the mean ± SD of three independent experiments. *P < 0.05

Fig. 5

Knockdown or blockade of SLC6A14 reduced CRC cell motility. A The migratory abilities of the HCT116 or Caco2 cells transfected with SLC6A14-siRNA or blockade of SLC6A14 with α-MT were evaluated by transwell chamber assays. Representative fields of migrated cells are shown. B Quantification of cells. C A wound healing assay was used to analyze the motility of the HCT116 or Caco2 cells transfected with SLC6A14-siRNA or blockade of SLC6A14 with α-MT. The data are expressed as the mean ± SD of three independent experiments. D The percent of cell migration was calculated by Image J and the data are shown.*P < 0.05

Fig. 6

Effects of SLC6A14 on CRC via the activation of mTOR signaling. A The protein expression of mTOR signaling and MMPs in the HCT116 or Caco2 cells transfected with SLC6A14-siRNA or blockade of SLC6A14 with α-MT was analyzed by Western blot. B The relative protein levels of SLC6A14 and mTOR signaling molecules, and β-actin was used as the internal control. C The relative protein levels of MMP2, MMP9, and MT1-MMP, and β-actin was used as the internal control. D Western blot analysis was used to determine the downstream effectors of mTOR in HCT116 or Caco2 cells transfected with SLC6A14-siRNA or blockade of SLC6A14 with α-MT. E The relative protein levels of P-p70S6, P-S6 and P-4EBP1, and β-actin was used as the internal control. F Western blot analysis was used to determine the total-Akt and phosphorylated-Akt (Ser473) in HCT116 or Caco2 cells transfected with SLC6A14-siRNA or blockade of SLC6A14 with α-MT. G The relative protein levels of Akt and P-Akt, and β-actin was used as the internal control. The data are expressed as the mean ± SD of three independent experiments. *P < 0.05

Fig. 7

Blockade of SLC6A14 with 2 mg/ml α-MT in vivo inhibits xenograft tumor growth. A The nude mice were sacrificed, and the tumor tissues were collected and photographed. B The tumor weights were measured. C The tumor growth curve was measured. D Immunohistochemical staining analysis of SLC6A14 and mTOR protein expression in the tumor tissues from each group, 1 bar = 50 μm. The data are expressed as the mean ± SD. *P < 0.05