Mutation SVCT2 promotes cell proliferation, invasion and migration in colorectal cancer

Abstract

The sodium-dependent vitamin C transporter 2 (SVCT2) surface glycoprotein regulates ascorbate accumulation in the plasma, often resulting in the induction of cancer cell death. Therefore, high expression of this gene associates with increased overall survival in several cancers. However, in colorectal cancer (CRC), high (likely mutated) SVCT2 expression relates to poor overall survival, and its functional significance has not been studied. Thus, we hypothesize that mutant SVCT2 expression could affect CRC patient survival. According to biological databases, SVCT2 has been found to be mutated frequently, and SVCT2 E264K has a particularly high pathogenic score (0.98), compared to other SVCT2 mutant sites, in CRC patients. Interestingly, our results reveal expression of SVCT2 E264K in many CRC tissues and cells. Also, we found wild-type SVCT2 expression to be largely localized to the cytoplasm and membrane, while SVCT2 E264K was restricted to the cytoplasm. We further found that SVCT2 E264K overexpression increases cell growth. By contrast, SVCT2 E264K knockdown significantly reduced cell proliferation and promoted cell apoptosis, resulting in inhibition of cell invasion and migration. Taken together, SVCT2 E264K plays a critical role in proliferation in CRC. Our results suggest that SVCT2 E264K could be a promising novel therapeutic target in CRC.

Keywords: SVCT2; cell proliferation; colorectal cancer; invasion; migration.

Figure 1

Overexpression of SVCT2 E264K in CRC. (A) Overall survival analysis showed that SVCT2 low expression (n=81) has a better prognosis than SVCT2 high expression(n=81). p=0.038, HR(high)=1.8, p(HR)=0.041. (B) Expression of SVCT2 E264K in 64 CRC patients using RT-PCR methods. (C) SVCT2 levels were confirmed in 10 human CRC cell lines. (D) Expression of SVCT2 E264K was analyzed by sequencing in SW620 and HCT-15. (E) SVCT2 localized to the membrane and cytosol in wild-type SVCT2-expressing SW620 cells , while HCT15 cells expressed SVCT2 E264K only in the cytosol.

Figure 2

The effects of SVCT2 E264K on cell proliferation. (A) Expression of p-Akt and Survivin was evaluated by western blotting in HEK293T cells. Cellular proliferation of SVCT2 WT and SVCT2 E264K-overexpressing cells analyzed using (B) trypan blue assay staining and (C) colony formation assay. *p<0.05 indicates significantly different from control group.

Figure 3

The effects of SVCT2 E264K knockdown on cell apoptosis. (A) Following SVCT2 E264K knockdown, as achieved by shRNA antagonism, HCT-15 cells were photographed under a light microscope. Scale bar, 100 μm. (B) Cell death after SVCT2 E264K knockdown was confirmed by (B) trypan blue staining and (C) FITC-annexin V and PI staining (D) caspase 3/7 activity. (E) Expression of p-Erk and p-Akt was shown by western blotting in HCT-15 cells. *p<0.05; significantly different from control group.

Figure 4

The effects of SVCT2 E264K knockdown on cell migration and invasion. (A) Migration of SVCT2 E264K knockdown HCT-15 cells was assessed using uncoated transwell chambers. (B) Invasion of SVCT2 E264K knockdown HCT-15 cells was confirmed with Matrigel™-coated transwell chambers. Scale bar: 100 μm. *p<0.05 indicate significantly different from control group.