HINT2 downregulation promotes colorectal carcinoma migration and metastasis

Abstract

Histidine triad nucleotide-binding 2 (HINT2), a member of the histidine triad proteins family, sensitizes cells to apoptosis in hepatocellular carcinoma. Here, we showed that HINT2 expression is lower in primary colorectal cancer (CRC) and metastasis tissues than in normal colorectal tissues, and that HINT2 abundance is inversely correlated with CRC tumor stage. Treating CRC cells with 5-aza-2'-deoxycytidine, a demethylating agent, upregulated HINT2, suggesting HINT2 downregulation is caused by methylation of the gene promoter. HINT2 downregulation increased tumor migration and invasion in vitro, promoted CRC cell metastasis in vivo, and increased expression of epithelial-to-mesenchymal transition (EMT) markers. Furthermore, HINT2 downregulation depended on hypoxia inducible factor (HIF)-2α-mediated transcriptional activation of zinc finger E-box-binding homeobox 1 (ZEB1). These results suggest that HINT2 downregulation promotes HIF-2α expression, which induces EMT and enhances CRC cell migration and invasion. HINT2 may thus a useful clinical indicator of CRC progression and metastasis risk.

Keywords: HIF-2α; HINT2; ZEB1; colorectal cancer; epithelial–mesenchymal transition.

Figure 1. Endogenous HINT2 expression in CRC tissues and cell lines

IHC staining of HINT2 in normal para-cancer (A and D), primary cancer (B and E), and metastasis tissues (C: lymph node MT, F: liver MT), and quantification (G) of the above. Arrows indicate liver metastasis and normal liver tissue, original magnification, ×200. (H) RT-qPCR analysis of HINT2 expression; data represent mean ± SD, n = 46 per group, *P < 0.05 H. RT-qPCR analysis of HINT2 expression; data represent mean ± SD, group 1 represents tissue without metastasis (n = 20), and group 2 represents tissue with metastasis (n = 26), *P < 0.05 (I). HINT2was detected in CRC cell lines via real-time PCR (J) and western blotting (K) Bar graph shows SW620 cell-normalized HINT2 expression in CRC cell lines. Hint2 was detected in Lovo and SW620 cells via real-time PCR after treatment with control (DMSO) or 5-aza-dC (L). Relative HINT2 protein intensity in cancer tissues relative to tumor stage (M) *P < 0.05, **P < 0.001 compared to SW620 cells. All data are representative of at least three independent experiments.

Figure 2. HINT2 downregulation is positively associated with CRC cell metastasis and invasion

HINT2 knockdown in SW480 cells was confirmed by western blotting (A). Wound healing showing SW480 cell migration (B and C) after 24-h incubation. Cells were photographed under a phase contrast microscope. Transwell assay showing SW480 cell migration (D and E) after 48-h incubation (×200 magnification). Results were plotted as the average number of migrated cells from six random microscope fields. Transwell assay showing SW480 cell invasion (F) and qualification (G) after 48-h incubation (×200 magnification). Results were plotted as the average number of invading cells from six random microscope fields. All data are representative of at least three independent experiments. **P < 0.01, ***P < 0.001 compared to controls.

Figure 3. HINT2 overexpression is negatively associated with CRC cell migration and invasion

HINT2 overexpression in SW620 cells was confirmed by western blotting (A). Wound healing assay showing SW620 cell migration (B and C) after 24-h incubation. Cells were photographed under a phase contrast microscope. Transwell assay showing SW620 cell migration (D and E) (×200 magnification) after 48-h incubation. Results were plotted as the average number of migrated cells from six random microscope fields. Transwell assay showing SW620 cell invasion (F) and qualification (G) (×200 magnification) after 48-h incubation. Results were plotted as the average number of invading cells from six random microscope fields. All data are representative of at least three independent experiments. **P < 0.01, ***P < 0.001 compared to controls.

Figure 4. HINT2 downregulation induced EMT in CRC cells

SW480 cells with and without HINT2 downregulation, photographed under a phase contrast microscope (A). A mesenchymal phenotype was observed in HINT2 knockdown cells. HT-29 and SW480 HINT2 knockdown cells were transfected with the CDH1 promoter vector, pGL-CDH1, and relative luciferase activation was detected (B). Data from control cells were set at 100%. RT-PCR analysis of EMT transcription factor, CDH1, in HT-29 and SW480 HINT2 knockdown cells compared with controls (C). Data from control cells were set at 100%. HINT2 and several EMT markers were detected by western blotting in HT-29 (D) and SW480 (E). HINT2-downregulated and control cells. HINT2-downregulated cells had increased CDH2 and vimentin expression and decreased CDH1 expression. GAPDH was used as the loading control. RT-PCR analysis of EMT transcription factors, such as SNAI1, SNAI2, ZEB1, ZEB2, TWIST1, and TWIST2 in HT-29 (F) and SW480 (G). HINT2-downregulated and control cells. Data from control cells were set at 100%. *P < 0.05, ***P < 0.001 compared to controls. All experiments are detected and analyzed in triplicates.

Figure 5. Molecular markers confirm the essential role of ZEB1 in HINT2-induced EMT through HIF-2α

siRNA-mediated ZEB1 knockdown in SW480 HINT2-downregulated cell lines was confirmed by western blotting (A) ZEB1 inhibition rescues the mesenchymal phenotypic change (B) and CDH1 transcriptional inhibition (C) caused by HINT2 downregulation. HINT2 knockdown or control SW480 and HT-29 cells were transfected si-ZEB1 or si-Con (control), then transfected with the CDH1 promoter vector, pGL-CDH1, and relative luciferase activation was detected. Data from control cells were set at 100%. Western blot analysis of HINT2, ZEB1, and EMT markers in SW480 control, HINT2-knockdown, and HINT2/ZEB1 double knockdown cells (D) HINT2 and ZEB1 double knockdown nearly rescued the effect of HINT2 silencing alone on EMT. Real-time PCR analysis of HIF-2α in HT-29 and SW480 control and HINT2-knockdown cells (E) HINT2 knockdown enhanced HIF-2α expression. Activation of plu567 or pluc567-mut in SW480 cells with or without HINT2 knockdown (n = 3 replicate experiments) (F) Transwell assay showing SW480 cell migration (G) and invasion (H) after 48-h incubation (×200 magnification). Results were plotted as the average number of migrated cells from six random microscope fields. *P < 0.05, ***P < 0.001 compared to controls.

Figure 6. HINT2 downregulation promotes CRC metastasis in vivo

SW480 cells with and without HINT2 knockdown were injected intrasplenically into female BALB/c NOD mice, and mice were killed six weeks later. Tumor islets were observed in livers via H&E staining. Typical views of liver presenting macroscopic metastases at week six (A) Left panel: SW480 without HINT2 knockdown; right panel: SW480 with HINT2 knockdown. Numbers of liver metastases and micrometastases after intrasplenic injection (B). H&E (C) and HINT2 (D) staining in mouse metastatic liver tumor tissues. Western blot analysis of HINT2 and ZEB1 in liver metastases from control (group 1) and HINT2-downregulated (group 2) SW480 cell-injected mice (E) All experiments were performed in triplicate.