Knockdown Proteomics Reveals USP7 as a Regulator of Cell-Cell Adhesion in Colorectal Cancer via AJUBA

Abstract

Ubiquitin-specific protease 7 (USP7) is implicated in many cancers including colorectal cancer in which it regulates cellular pathways such as Wnt signaling and the P53-MDM2 pathway. With the discovery of small-molecule inhibitors, USP7 has also become a promising target for cancer therapy and therefore systematically identifying USP7 deubiquitinase interaction partners and substrates has become an important goal. In this study, we selected a colorectal cancer cell model that is highly dependent on USP7 and in which USP7 knockdown significantly inhibited colorectal cancer cell viability, colony formation, and cell-cell adhesion. We then used inducible knockdown of USP7 followed by LC-MS/MS to quantify USP7-dependent proteins. We identified the Ajuba LIM domain protein as an interacting partner of USP7 through co-IP, its substantially reduced protein levels in response to USP7 knockdown, and its sensitivity to the specific USP7 inhibitor FT671. The Ajuba protein has been shown to have oncogenic functions in colorectal and other tumors, including regulation of cell-cell adhesion. We show that both knockdown of USP7 or Ajuba results in a substantial reduction of cell-cell adhesion, with concomitant effects on other proteins associated with adherens junctions. Our findings underlie the role of USP7 in colorectal cancer through its protein interaction networks and show that the Ajuba protein is a component of USP7 protein networks present in colorectal cancer.

Keywords: AJUBA; USP7; cancer; deubiquitinase; inducible knock-down.

Graphical abstract

Fig. 1

LS180 is an effective model for investigating USP7 function in colorectal cancer.A, scatterplot comparing the distribution of USP7 gene effect processed scores (CERES, DEMETER2) between the RNAi dataset (y) and CRISPR dataset (x). Selected cell-lines with the largest USP7 gene-effect scores from RNAi and CRISPR are labeled in addition to DLD1, an additional CRC cell-line used in this study. All analysis performed in DepMap portal (depmap.org). B, efficient degradation of USP7 at the indicated time points. LS88 colon carcinoma cells stably carrying a pTER-USP7 shRNA construct and a Tet repressor (TR)-construct were treated with 1 μg/ml doxycycline for 24, 48, and 72 h and examined by Western blotting for reduction of endogenous USP7; GAPDH was used as a loading control. C, LS88 cells (with inducible siRNA targeting USP7) were treated with 1 μg/ml concentrations of doxycycline for 72 h before being seeded in 6-well plates and incubated for 7 to 14 days. Graph represents mean ± SD (two tailed, unpaired t test), p ≤ 0.0001.

Fig. 2

Proteomic analysis of LS88 with/without doxycycline treatment for USP7 knockdown.A, principal component analysis scores plot showing the clustering of proteins according to their distributions in different groups. Control LS88 represents cells at hour zero (0 h), LS88 treated with doxycycline for 72 h (72 h + doxycycline) or left without treatment for 72 h (72 h). B, heatmap of normalized intensity values (light/low – dark/high) for each group (0 h untreated; 72 h untreated; 72 h + doxycycline). All proteins (N = 444) with p < 0.05 for treated versus untreated (at 72 h) are included. C, volcano plot showing differentially responsive proteins in comparison of 72 h untreated versus 72 h doxycycline-treated cells. Selected proteins from enriched pathway and process categories are labeled. D, annotation analysis of the sets of proteins (p < 0.05) decreased following doxycycline treatment (after 72 h). Pathways from DAVID tool are sorted by p value and the top 15 most significantly enriched pathways shown. Ubiquitin-mediated proteolysis category is indicated by arrow. E, the top 15 most significantly decreased proteins (p < 0.05) following doxycycline treatment (after 72 h) that are involved in protein polyubiquitination. TRIM27, a USP7 protein-complex partner, is the most significantly altered protein following USP7 knockdown in this category (Analysis performed in Metascape). Proteins that are known to be direct substrates or interaction partners of USP7 (according to STRING) are indicated with arrow. F, dendrogram showing significant Gene Ontology cellular components for the set of proteins (p < 0.05) decreased following doxycycline treatment. Enrichment p-values are indicated and only GO terms with FDR p-value <0.05 were included. GO terms associated with cell-cell junction functions are highlighted with orange bar. Dendrogram circle shape size indicates p-value.

Fig. 3

Western blot analysis of Ajuba abundance following USP7 knockdown.A, total cell lysate treated with doxycycline in different time point were collected and analysis using immunoblotting. B, LS88 cells treated or untreated with doxycycline were examined on Ajuba protein expression. Unpaired Student’s t test was used for three independent experiments. p < 0.0001. GAPDH was used as a loading control. C, total cell lysates from LS88 and (D) HCT116 treated with FT671 (10 μM) for indicated times. GAPDH is a loading control.

Fig. 4

Immunofluorescence analysis of Ajuba levels following USP7 knockdown in LS88 cells.A, immunofluorescence staining of Ajuba, (B) USP7 (green) in LS88 cells treated with doxycycline (DOX+) or untreated (DOX−). Cells were fixed and stained with DAPI (blue) to stain nuclei. Scale bars represents 30 μm. C, data were corrected by subtracting local background values from cytoplasm and nucleus mean gray values. Statistical differences were determined by Unpaired t test of percentage change of Ajuba and USP7 in cytoplasm and nuclei in LS88 cells. Data are mean ± SD.

Fig. 5

USP7 interacts with Ajuba and Ajuba knockdown does not affect USP7 protein levels in CRC cells.A, mRNA levels of USP7 in doxycycline-treated (DOX+) or untreated (DOX-) LS88 cells. ∗∗p < 0.01. Data are mean ± SD. B, mRNA levels of Ajuba in DOX + or DOX- LS88 cells. Unpaired t-tests were used for analysis in order to assess any differences between the means. Experiments were performed in triplicate, n = 3. C, LS88 cells total lysates were immunoprecipitated with anti USP7 antibody or IgG, and Ajuba was analyzed using indicated antibodies in LS88 cells. D, LS88 and LS174T cells expressing Ajuba, control siRNA, or Ajuba siRNA were analyzed by Western blotting and USP7 protein expression were examined using the indicated antibodies. As a loading control, GAPDH protein levels were assessed.

Fig. 6

Mechanical stress analysis of cell-cell adhesion in LS88 cells.A, monolayer cultures untreated or treated with siControl or siUSP7 were treated with dispase and incubated until the monolayer was detached from the culture surface, followed by shaking to induce fragmentation. The fragments were counted for all three conditions and the bar graph shows the average number of fragments. One-way ANOVA was used. ∗∗∗∗p < 0.0001. Mean ± SD from three independent experiments. Arrow indicates increased fragmentation of monolayers following mechanical stress. Scale bar represents 5 mm. B, monolayer cultures untreated or treated with siControl or siAJuba were treated with dispase and incubated until the monolayer was detached from the culture surface, followed by shaking to induce fragmentation. The fragments were counted for all three conditions and the bar graph shows the average number of fragments. One-way ANOVA was used. ∗∗∗∗p < 0.0001. Mean ± SD from three independent experiments. Arrow indicates increased fragmentation of monolayers following mechanical stress. Scale bar represents 5 mm.

Fig. 7

USP7 knockdown and effects on cadherin proteins and their substrates.A, protein expression of N-cadherin and E-cadherin in LS88 cells treated with doxycycline (DOX) for the indicated time. GAPDH was used as a loading control. Adding doxycycline induces USP7 expression in LS88 cells. B, protein expression of N-cadherin and E-cadherin in HCT116 cells transfected with siUSP7 or siControl or left untreated. GAPDH was used as a loading control. C, α-catenin and γ-catenin expression were analyzed by Western blot. N = 3. GAPDH was used as a loading control. D, cell lysates from LS88 cells were immunoprecipitated with USP7; IgG antibodies followed by immunoblotting to examine the impact of USP7 knockdown on E-cadherin and γ-catenin (JUP).

Fig. 8

USP7 knockdown and effects on desmosome and hemidesmosome protein levels.A, protein expression of ITGA6, (B) ITGB4, and (C) DSG2 were analyzed using Western blot. GAPDH was used as a loading control. D, LS88 cells were transfected with siControl and siAJUBA or left untreated; the cell lysates were subjected to Western blotting with indicated antibodies and graph (E) showing protein levels of JUP in the three conditions. The ordinary one-way-ANOVA was used from three independent experiments. The significant difference between the two groups untreated and siAjuba groups is ∗p < 0.05. Mean ± SD. F, Ajuba protein was knocked down in LS174T using siRNA and Western blot was performed using AJUBA and JUP antibodies. G and H, cell lysates from LS88 cell were subjected to immunoblotting with AJUBA and α -catenin and N-cadherin antibodies. GAPDH was used as a loading control.