Unspliced XBP1 contributes to cholesterol biosynthesis and tumorigenesis by stabilizing SREBP2 in hepatocellular carcinoma

Abstract

Cholesterol biosynthesis plays a critical role in rapidly proliferating tumor cells. X-box binding protein 1 (XBP1), which was first characterized as a basic leucine zipper-type transcription factor, exists in an unspliced (XBP1-u) and spliced (XBP1-s) form. Recent studies showed that unspliced XBP1 (XBP1-u) has unique biological functions independent from XBP1-s and could promote tumorigenesis; however, whether it is involved in tumor metabolic reprogramming remains unknown. Herein, we found that XBP1-u promotes tumor growth by enhancing cholesterol biosynthesis in hepatocellular carcinoma (HCC) cells. Specifically, XBP1-u colocalizes with sterol regulatory element-binding protein 2 (SREBP2) and inhibits its ubiquitination/proteasomal degradation. The ensuing stabilization of SREBP2 activates the transcription of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), a rate-limiting enzyme in cholesterol biosynthesis. We subsequently show that the XBP1-u/SREBP2/HMGCR axis is crucial for enhancing cholesterol biosynthesis and lipid accumulation as well as tumorigenesis in HCC cells. Taken together, these findings reveal a novel function of XBP1-u in promoting tumorigenesis through increased cholesterol biosynthesis in hepatocarcinoma cells. Hence, XBP1-u might be a potential target for anti-tumor therapeutic strategies that focus on cholesterol metabolism in HCC.

Keywords: Cholesterol biosynthesis; SREBP2; Tumorigenesis; Unspliced XBP1 (XBP1-u); XBP1.

Fig. 1

XBP1-u regulates HCC cell cholesterol level. A, B Accumulation of lipid droplets in XBP1-silenced HCC cells, as determined using Nile Red staining. Representative images (A; scale bars: 200 μm) and quantification results (B; n = 6) are shown. C Total cholesterol level in XBP1-silenced HCC-cells. D LDL level in XBP1-silenced HCC-cells. E–F Accumulation of lipid droplets in XBP1-u-overexpressed HCC-cells, as determined using Nile Red staining. Representative images (E; scale bars: 200 μm) and quantification results (F; n = 6) are shown. G. Total cholesterol level in XBP1-u-overexpressed HCC-cells. H LDL level in XBP1-u-overexpressed HCC-cells. Cells transfected with shCon or pcCon were used as controls. Total protein was used for normalization of total cholesterol and LDL levels. Quantification data are shown as mean ± SD (n = 3; unless further indicated). pcCon pcDNA3.1(+); **P < 0.01

Fig. 2

Cholesterol is crucial for XBP1-u regulation on HCC cell tumorigenic potential. A, B Proliferation potential of XBP1-silenced (A) and XBP1-u-overexpressed (B) HCC-LM3 cells, as determined using EdU-incorporation assay. Representative images (left; scale bars: 200 μm) and ratio of proliferative cells (right; n = 6) are shown. C, D Colony formation potential of XBP1-silenced (C) and XBP1-u-overexpressed (D) HCC-LM3 cells. Representative images (left) and the number of the colonies formed (right; n = 6) are shown. E Viability of XBP1-silenced HCC-LM3 cells treated with cholesterol (final concentration: 10 μg/mL) at indicated time points (n = 3). F, G Proliferation potential of XBP1-silenced HCC-LM3 cells treated with cholesterol (final concentration: 10 μg/mL), as determined using EdU-incorporation assay. Representative images (F; scale bars: 200 μm) and ratio of proliferative cells (G; n = 6) are shown. H Colony formation potential of XBP1-silenced HCC-LM3 cells treated with cholesterol (final concentration: 10 μg/mL). Representative images (left) and the number of the colonies formed (right; n = 6) are shown. Cells transfected with shCon or pcCon were used as controls. Quantification data are shown as mean ± SD. pcCon, pcDNA3.1(+); *P < 0.05; **P < 0.01

Fig. 3

Inhibition of cholesterol biosynthesis suppresses XBP1-u-mediated HCC cell tumorigenic potential. A Accumulation of lipid droplets in XBP1-u-overexpressed HCC-LM3 cells treated with statin (final concentration: 15 μM), as analyzed using Nile Red staining. Representative images (left; scale bars: 200 μm) and quantification results (right; n = 6) are shown. B, C Total cholesterol (B) and LDL (C) levels in XBP1-u-overexpressed HCC-LM3 cells treated with statin (final concentration: 15 μM). D Viability of XBP1-u-overexpressed HCC-LM3 cells treated with statin at indicated time points (final concentration: 15 μM). E, F Proliferation potential of XBP1-u-overexpressed HCC-LM3 cells treated with statin (final concentration: 15 μM), as determined using EdU-incorporation assay. Representative images (E; scale bars: 200 μm) and ratio of proliferative cells (F; n = 6) are shown. G Colony formation potential of XBP1-u-overexpressed HCC-LM3 cells treated with statin (final concentration: 15 μM). Representative images (left) and the number of the colonies formed (right; n = 6) are shown. Cells transfected with pcDNA3.1(+) and treated with DMSO were used as controls. Quantification data are shown as mean ± SD (n = 3, unless further indicated). *P < 0.05; **P < 0.01

Fig. 4

XBP1-u regulates HMGCR expression in HCC cells at its transcriptional level. A, B HMGCR mRNA expression level in XBP1-silenced (A) and XBP1-u-overexpressed (B) HCC-LM3, MHCC-97H, and HepG2 cells, as analyzed using qRT-PCR. C, D HMGCR protein expression level in XBP1-silenced (C) and XBP1-u-overexpressed (D) HCC-LM3, MHCC-97H, and HepG2 cells, as analyzed using western blotting. Cells transfected with shCon or pcCon were used as controls. β-actin was used for qRT-PCR normalization and as western blotting loading control. Quantification data are shown as mean ± SD (n = 3). pcCon, pcDNA3.1(+); **P < 0.01

Fig. 5

HMGCR is crucial for XBP1-u regulation on cholesterol biosynthesis and tumorigenic potential of HCC cells. A Protein expression level of HMGCR in XBP1-silenced, HMGCR-overexpressed HCC-LM3 cells, as analyzed using western blotting. B, C Accumulation of lipid droplets in XBP1-silenced, HMGCR-overexpressed HCC-LM3 cells, as analyzed using Nile Red staining. Representative images (B; scale bars: 200 μm) and quantification results (C; n = 6) are shown. D, E Total cholesterol and LDL levels in XBP1-silenced, HMGCR-overexpressed HCC-LM3 cells. F Viability of XBP1-silenced, HMGCR-overexpressed HCC-LM3 cells at indicated time points. G, H Proliferation potential of XBP1-silenced, HMGCR-overexpressed HCC-LM3 cells, as determined using EdU-incorporation assay. Representative images (G; scale bars: 200 μm) and ratio of proliferative cells (H; n = 6) are shown. I Colony formation potential of XBP1-silenced, HMGCR-overexpressed HCC-LM3 cells. Representative images (left) and the number of the colonies formed (right; n = 6) are shown. Cells transfected with shCon and pcEF9-Puro were used as controls. β-actin was used as western blotting loading control. Total protein was used for normalization of total cholesterol and LDL levels. Quantification data are shown as mean ± SD (n = 3, unless further indicated). *P < 0.05; **P < 0.01

Fig. 6

XBP1-u stabilizes SREBP2 protein by inhibiting its ubiquitin-proteasomal degradation. A, B SREBP2 protein expression level in XBP1-silenced (A) and XBP1-u-overexpressed (B) HCC-LM3 cells, as analyzed using western blotting. C HMGCR and SREBP2 protein expression levels in SREBP2-silenced, XBP1-u-overexpressed HCC-LM3 cells, as analyzed using western blotting. D Colocalization of endogenous SREBP2 and XBP1-u, as determined by immunofluorescence staining using anti-SREBP2 and anti-XBP1-u antibodies (scale bars: 20 μm). E Physical interaction between XBP1-u and SREBP2, as determined using anti-XBP1-u immunoblotting of cell lysate immunoprecipitated with anti-SREBP2 antibody and vice versa. F, G Degradation rates of SREBP2 protein in XBP1-u-overexpressed HCC-LM3 cells, as analyzed using western blotting at indicated time points after the addition of cycloheximide (final concentration: 200 μg/ml). Western blotting result (F) and the half-life of SREBP2 protein (G) are shown. H SREBP2 protein expression level in XBP1-silenced HCC-LM3 cells treated with MG132 (final concentration: 20 μM) for 8 h, as analyzed using western blotting. I, J SREBP2 ubiquitination levels in HCC-LM3 cells transfected with pcSREBP2, pcUbi, and shXBP1 (I) or pcXBP1-u (J), as analyzed using anti-ubiquitin immunoblotting of cell lysates immunoprecipitated with anti-SREBP2 antibody. K SREBP2 and HMGCR protein expression levels in HCC-LM3 cells transfected with FLAG-XBP1-u, FLAG-XBP1-u-N and FLAG-XBP1-u-C, as determined using western blotting. L SREBP2 ubiquitination level in HCC-LM3 cells transfected with pcFLAG-XBP1-u-C, pcSREBP2, and pcUbi, as analyzed using anti-ubiquitin immunoblotting of cell lysates immunoprecipitated with anti-SREBP2 antibody. Cells transfected with shCon and/or pcCon, or pcFlag, were used as controls. β-actin was used as western blotting loading control. Quantification data are shown as mean ± SD (n = 3). pcCon, pcDNA3.1(+); pcSREBP2: full-length SREBP2 overexpression vector; IB immunoblotting; IP immunoprecipitation; Ubi ubiquitination; **P < 0.01

Fig. 7

SREBP2 is critical for XBP1-u-induced cholesterol biosynthesis and tumorigenic potential of HCC cells. A Protein expression levels of SREBP2 and HMGCR in XBP1-silenced, SREBP2-overexpressed HCC-LM3 cells, as analyzed using western blotting. B, C Accumulation of lipid droplets in XBP1-silenced, SREBP2-overexpressed HCC-LM3 cells, as analyzed using Nile Red staining. Representative images (B; scale bars: 200 μm) and quantification results (C; n = 6) are shown. D, E Total cholesterol (D) and LDL (E) levels in XBP1-silenced, SREBP2-overexpressed HCC-LM3 cells overexpressing SREBP2. F Viability of XBP1-silenced, SREBP2-overexpressed HCC-LM3 cells at indicated time points. G, H Proliferation potential of XBP1-silenced, SREBP2-overexpressed HCC-LM3 cells, as determined using EdU-incorporation assay. Representative images (G; scale bars: 200 μm) and ratio of proliferative cells (H; n = 6) are shown. I Colony formation potential of XBP1-silenced, SREBP2-overexpressed HCC-LM3 cells. Representative images (left) and numbers of the colonies formed (right; n = 6) are shown. Cells transfected with shCon and pcEF9-Puro were used as controls. β-actin was used as western blotting loading control. Total protein was used for normalization of total cholesterol and LDL levels. Quantification data are shown as mean ± SD (n = 3, unless further indicated). pcSREBP2 full-length SREBP2 overexpression vector; *P < 0.05; **P < 0.01

Fig. 8

XBP1-u mediates hepatocarcinogenesis potential by positively regulates SREBP2. A Protein expression levels of SREBP2 and HMGCR in HCC-LM3/shCon (shCon), HCC-LM3/shXBP1 (shXBP1) and HCC/shXBP1/pcSREBP2 (shXBP1/pcSREBP2) cells, as analyzed using western blotting. B–D Tumorigenesis potentials of HCC-LM3/shCon, HCC-LM3/shXBP1, and HCC-LM3/shXBP1/pcSREBP2 cells, as determined in vivo by subcutaneous injection of these cells into Balb/c-nu/nu mice (n = 6). Tumor volumes at the indicated time points (B), morphological images (C) and tumor weight (D) at day 16 after transplantation are shown. E XBP1-u, SREBP2, and HMGCR protein expression levels in the xenografted tumors formed by the indicated cells, as determined using western blotting. F Immunohistochemistry staining using anti-SREBP2, anti-XBP1-u, and anti-HMGCR antibodies showing the expression levels of SREBP2, XBP1-u, and HMGCR in tissue sections of xenografted tumors in BALB/c-nu/nu mice injected with the indicated cell lines (scale bars: 50 μm). G Accumulation of lipid droplets in the tissue section of xenografted tumors in BALB/c-nu/nu mice injected with the indicated cell lines (scale bars: 50 μm). H, I Total cholesterol (H) and LDL (I) levels in the xenografted tumors in BALB/c-nu/nu mice injected with the indicated cell lines (n = 3). J Schematic diagram showing the mechanism of XBP1-u regulation on the SREBP2/HMGCR axis. β-actin was used as western blotting loading control. Quantification data are shown as mean ± SD. **P < 0.01