Thrombospondin-1 counteracts the p97 inhibitor CB-5083 in colon carcinoma cells

Abstract

p97 has recently emerged as a therapeutic target for cancer due to its essential functions in protein homeostasis. CB-5083 is a first-in-class, potent and selective ATP-competitive p97 inhibitor that induces proteotoxic stress in cancer cells. Potential mechanisms regulating the sensitivity of cells to p97 inhibition remain poorly studied. Here, we demonstrate that Thrombospondin-1 (THBS1) is a CB-5083-upregulated gene that helps confer resistance of HCT116 cells to CB-5083. Our immunoblotting and immunofluorescence data showed that CB-5083 significantly increases the steady-state abundance of THBS1. Blockade of THBS1 induction sensitized cells to CB-5083-mediated growth inhibition. Suppression of THBS1 caused an increase of CB-5083-induced sub-G1 population and caspase 3/7 activity suggesting that its function is linked to the survival of cancer cells in response to p97 inhibition. Altogether our data provide new evidence that THBS1 is important for the susceptibility of cells to p97 inhibition.

Keywords: Thrombospondin-1; cb-5083; p97; protein homeostasis.

Figure 1.

Identification and validation of genes upregulated by the p97 inhibitor CB-5083. (A) Summary of the GEO dataset evaluating whole transcriptome in HCT116 cells in response to CB-5083 (GSE73588). Independent t-test was performed to calculate Log2 ratios of CB-5083/DMSO treatments, with P < 0.0015. (B) HCT116 cells were treated with 1 μM CB-5083 for 8 h. Relative expressions of the indicated genes were analyzed by RT-PCR and quantified. p < 0.05 for differential gene expression. Data represent the mean (N = 3) and SD error.

Figure 2.

The steady-state level of THBS1 protein is increased by CB-5083. (A) HCT116 cells were treated with the indicated concentrations of CB-5083, Bortezomib and MLN4924 for 8 h. The steady-state levels of THBS1 and indicated proteins were analyzed by immunoblotting. Actin was used as a loading control. (B) Relative abundance of THBS1 protein was quantified (N = 3, SD error). (C) Relative abundance of known biomarkers of p97 inhibition (HERP1, CHOP and ATF4) was quantified (N = 3, SD error). (D) Mean fold changes (N = 3, SD error) for CDT1 and p21 with 3 μM CB-5083, 1 μM Bortezomib and 3 μM MLN4924. (E) An immunofluorescence assay for THBS1 (green), Actin (red) and DAPI (blue) in HCT116 cells treated with or without 1.5 μM CB-5083 for 8 h (N = 3, SD error).

Figure 3.

Knockdown of THBS1 sensitizes cells to CB-5083 and promotes apoptosis. (A) HCT116 cells transfected with siControl or siTHBS1-1 (shortly siTHBS1) were treated with or without 0.75 μM CB-5083 for 8 h. Total extracts were analyzed by immunoblotting. Actin was used as a loading control. (B) HCT116 cells transfected with siControl or siTHBS1 were treated with or without 0.375 μM CB-5083 for 72 h. Cells stained with crystal violet and are shown. (N = 3, SD error). *p < 0.05. (C) HCT116 cells transfected with siControl or siTHBS1 were treated with or without 0.375 μM CB-5083. Cell numbers were counted at 24 h intervals (N = 3, SD error). *p < 0.05. (D) HCT116 cells transfected with siControl or siTHBS1 were treated with indicated concentrations of CB-5083. Cell viability was analyzed by quantifying cellular ATP levels (N = 3, SD error). *p < 0.05. (E) HCT116 cells transfected with siControl or siTHBS1 were treated with indicated concentrations of CB-5083 for 72 h. Cell cycle was analyzed by flow cytometry and the percent Sub-G1 is shown (N = 3, SD error). *p < 0.05. (F) HCT116 cells transfected with siControl or siTHBS1 were treated with indicated concentrations of CB-5083 for 72 h. Caspase 3/7 activities were measured by immunofluorescence (N = 3, SD error). *p < 0.05.