SUMO1 degrader induces ER stress and ROS accumulation through deSUMOylation of TCF4 and inhibition of its transcription of StarD7 in colon cancer

Abstract

Small molecule degraders of small ubiquitin-related modifier 1 (SUMO1) induce SUMO1 degradation in colon cancer cells and inhibits the cancer cell growth; however, it is unclear how SUMO1 degradation leads to the anticancer activity of the degraders. Genome-wide CRISPR-Cas9 knockout screen has identified StAR-related lipid transfer domain containing 7 (StarD7) as a critical gene for the degrader's anticancer activity. Here, we show that both StarD7 mRNA and protein are overexpressed in human colon cancer and its knockout significantly reduces colon cancer cell growth and xenograft progression. The treatment with the SUMO1 degrader lead compound HB007 reduces StarD7 mRNA and protein levels and increases endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) production in colon cancer cells and three-dimensional (3D) organoids. The study further provides a novel mechanism of the compound anticancer activity that SUMO1 degrader-induced decrease of StarD7 occur through degradation of SUMO1, deSUMOylation and degradation of T cell-specific transcription 4 (TCF4) and thereby inhibition of its transcription of StarD7 in colon cancer cells, 3D organoids and patient-derived xenografts (PDX).

Keywords: ER stress; SUMO1 degrader; StarD7; TCF4; colon cancer.

FIGURE 1

StarD7 is overexpressed in colon cancer and required for the cancer growth. (A,B) The expression of StarD7 mRNA in different types of human cancers (A) and colon cancer (B) as compared with matched normal tissues from TCGA and GTEx cohorts (*, P<0.05; **, P<0.01; ***, P<0.001). (C,D) The expression of StarD7 protein in several types of human cancer (C) and colon cancer (D) and matched normal colon tissues from CPTAC dataset. (E) Human colon cancer tissue and matched adjacent normal tissue were analyzed by western blots for StarD7 protein levels. (F) CRISPR-Cas9 knockout of StarD7 (sgStarD7) and sgRNA control (sgCont) HCT116 clones were analyzed by western blots for StarD7 protein levels with β-actin as the loading control. (G) StarD7 knockout clones (sgStarD7#7 and #15) and sgCont clones were analyzed for cell growth by cell number counting (n=3; ***P<0.001 by Student’s t-test). (H) HCT116 sgStarD7 and sgCont clones (5×10⁶) were inoculated subcutaneously into mice (+/− SEM, n=8 per group, **p<0.01; Wilcoxon test). (I) The survival of the mice bearing HCT116 sgStarD7 and sgCont clones was evaluated by Kaplan Meier survival curve (*** p<0.001). (J) The xenografts were removed and analyzed by western blots for StarD7 protein levels with β-actin as the loading control in the xenografts generated from sgStarD7 and sgCont HCT116 clones. Ns, not significant; TCGA, the Cancer Genome Atlas.

FIGURE 2

HB007 treatment leads to StarD7 downregulation and ER stress. (A) HCT116 and DLD1 cells were treated with HB007 at the indicated concentrations for 48 h and examined by western blots for StarD7 and GRP78/BIP levels (upper panel). β-actin was used as the protein loading control and the molecular weight were indicated (left). The treated cells were also analyzed by RT-PCR for XBP1 mRNA splicing from the band of 441 bp to 416 bp. GAPDH was used as a loading control (lower panel). (B) HCT116 cells were treated with 4 μM of HB007 for 24 h and analyzed for phospholipid accumulation by Oil red O-staining (points: n=6; ***p<0.001; Student’s t-test). (C) HCT116 and DLD1 cells were treated with HB007 at the indicated concentrations for 24 h and analyzed for ROS levels (points: n=6; ***p<0.001, Student’s t-test). (D-E) HCT116 (D) and DLD1 cells (E) were treated with DMSO or HB007 at the indicated concentrations for 15 days and analyzed for colony number and colony diameter (means +/−S.D; n=3; ** p<0.01 and ***p<0.001; one way ANOVA and Turkey’s multiple comparisons test). (F) 3D organoids were treated with DMSO or HB007 at the indicated concentrations for 5 days (upper panel) and analyzed by western blots for StarD7 protein levels (bottom panel). ROS, reactive oxygen species.

FIGURE 3

HB007 activity is dependent of StarD7. (A,B) HCT116 sgStarD7 and sgCont clones were treated with HB007 for 3 days at the indicated concentrations, stained with crystal violet (A) and analyzed for cell growth by cell viability assay (B; means +/−S.D; n=6; **** p<0.0001; Student’s t-test). (C) HCT116 sgStarD7 and sgCont clones were treated with HB007 at the indicated concentrations (upper panel) for 48 h and analyzed by western blots using the antibodies indicated (left) with β-actin as the loading control. The treated cells were analyzed for XBP1 splicing by RT-PCR with GAPDH as the loading control (lower panel). (D) HCT116 sgStarD7 and sgCont clones were treated with 4 μM of HB007 for 24 h and then stained with Oil red O (left panel); the phospholipid accumulation was measured (right panel; means +/−S.D; n=6; **** p<0.0003; Student’s t-test). (E) HCT116 sgStarD7 and sgCont clones were treated with DMSO or HB007 at 4 μM for 24 h and analyzed for the intracellular ROS levels (means +/−S.D; n=6; **** p<0.0001; Student t-test). ROS, reactive oxygen species.

FIGURE 4

HB007 reduces StarD7 through SUMO1 E3 ligase. (A) SUMO1 knockout (sgSUMO1) and control (sgCont) clones were treated with HB007 at 4 μM for 48 h and analyzed by western blots for StarD7 and GRP78/BIP protein levels. (B) SUMO1 knockout (sgSUMO1) and control (sgCont) HCT116 clones were treated with HB007 at 4 μM for 24 h and analyzed for intracellular ROS levels (means +/−S.D; n=6; **** p<0.0001; Student’s t-test). (C) CAPRIN1 knockout (sgCAPRIN1) and control (sgCont) HCT116 clones were treated with HB007 for 48 h at the indicated doses and examined by western blots for StarD7 protein levels (upper panel) and by RT-PCR for XBP1 splicing (lower panel). (D) HCT116 sgCAPRIN1 and sgCont clones treated with HB007 at 4 μM for 24 h and tested for intracellular ROS levels (means +/−S.D; n=6; **** p<0.0001; Student’s t-test). ROS, reactive oxygen species.

FIGURE 5

HB007 inhibits StarD7 transcription. (A) HCT116 cells were treated with HB007 for 24 h, then MG132 or Bafilomycin A1 at the indicated concentrations for 16 h and analyzed by western blots for StarD7 protein levels. (B) HCT116 and DLD1 were treated with HB007 at the indicated concentrations for 24 h and analyzed by RP-PCR for StarD7 mRNA levels. (C) HCT116 cells were co-transfected with YFP-SUMO1-GV or YFP-SUMO1-GG together with Flag-TCF4 or empty vector as a control and subjected to Flag immunoprecipitation and western blots for TCF4-sumoylation detection using GFP and TCF4 antibodies as indicated (left). (D-E) HCT116 (D) and sgSUMO1 and sgCont cells (E) were infected with TCF/LEF Luciferase Reporter Lentivirus and treated with HB007 at the indicated concentrations for 24 h, followed by Wnt3a to induce the signaling cascade and luciferase signal (means +/−S.D; n=6; ** p<0.009 *** p<0.0001, *** p<0.0005; Student’s t-test).

FIGURE 6

HB007 reduces StarD7 gene transcription through induction of TCF4 protein degradation. (A-B) HCT116 cells (A) and 3D organoids (B) were treated with HB007 at the indicated concentrations for 48 h and analyzed by western blots for TCF4 protein levels with β-actin as the loading control. (C) HCT116 sgSUMO1 and sgCont clones were treated with 4 μM of HB007 for 48 h and examined by western blots for TCF4 protein levels. (D) HCT116 were transfected with increasing amount of YFP-SUMO1 plasmid as indicated (top) for 48 h and analyzed by western blots for TCF4 and StarD7 protein levels and YFP-SUMO1 conjugated proteins using the antibodies as indicated (left). (E) HCT116 cells were treated with HB007 at 2 μM for 24 h and MG132 at 1 or 2 μM for the last 4 h; TCF4 expression was examined by western blots with β-actin as the loading control.

FIGURE 7

HB007 reduces SUMO1 and StarD7 protein and suppresses PDX xenograft progression. (A) Spearman correlation between the expression level (TPM) of SUMO1 and StarD7 gene in colon cancer. (B) Colon cancer PDX mice were treated with HB007 (50 mg/kg) for 15 days beginning after a week of tumor inoculation. The xenograft sizes were measured for 15 days with the data presented on the upper panel) at means ± SEM (n = 6 per group, ***p <0.00009 by Wilcoxon test) and representative images of colon xenografts at the end of the treatment were presented on the bottom panel. (C) Colon cancer PDX mice were treated with the indicated doses of HB007 through intraperitoneal injection once per day for 3 days beginning after a week of tumor inoculation; xenograft tissues were analyzed by western blotting for StarD7 protein (top panel) and dot blotting for total levels of SUMO1 with SUMO2/3 (middle panel) with the densities quantified (bottom panel). (D) IHC was carried out using Ki-67 antibody on the vehicle (top panel) and HB007 treated xenografts (bottom panel) with the photos were taken under 10×20 magnification. (E) Ki-67 positive cells were counted under 10×40 magnification with the data presented at means ± SEM (n = 10 per group, ****p<0.0001). (F) IHC for PARP was carried out on the vehicle (top panel), HB007 treated xenografts (middle panel) and normal human lymph node (bottom panel) as the positive control. (G) Graphical illustration of the mechanisms of the drug action of SUMO1 degrader. IHC, immunohistochemistry; PDX, patient-derived xenograft.