The deubiquitinase USP54 is overexpressed in colorectal cancer stem cells and promotes intestinal tumorigenesis

Abstract

Ubiquitin-Specific Proteases (USPs) are deubiquitinating enzymes frequently deregulated in human malignancies. Here, we show that USP54 is overexpressed in intestinal stem cells and demonstrate that its downregulation in colorectal carcinoma cells impedes tumorigenesis. We have generated mutant mice deficient for this deubiquitinase, which are viable and fertile, and protected against chemically-induced colorectal carcinoma. Furthermore, we show that USP54 is upregulated in human colon cancer and associates with poor prognosis. In agreement with these results, Usp54 downregulation in mouse melanoma cells inhibits lung metastasis formation. Collectively, this work has uncovered the pro-tumorigenic properties of USP54, highlighting the importance of deubiquitinating enzymes as promising targets for the development of specific anti-cancer therapies.

Keywords: CSCs; degradome; gene targeting; ubiquitin-specific proteases.

Figure 1. USP54 is upregulated in intestinal stem cells and induces colorectal carcinoma progression

A. Analysis of USP54 expression from microarray data (GEO accession number GSE27605). High, high Lgr5 or EphB2 receptor expression levels; Med, medium EphB2 receptor expression levels; Low, low Lgr5 or EphB2 receptor expression levels. B. qRT-PCR analysis of USP54 expression in two different colorectal carcinoma xenografts (SP5 and SP9), two-tailed Student's t-test (***, P < 0.001). C. Anchorage-independent growth of control (pLKO.1) and USP54-depleted HCT116 cells (shUSP54.854 and shUSP54.856), two-tailed Student's t-test (**, P < 0.01; ***, P < 0.001). RFU, relative fluorescence units. D. qRT-PCR analysis of USP54 expression in HCT116 cells transduced with control (pLKO.1) or USP54-specific shRNAs (shUSP54.854 and shUSP54.856). Statistical significance was assessed by two-tailed Student's t-test (***, P < 0.001). E. MTT proliferation analysis of wild-type and USP54-deficient HCT116 cells, Mann Whitney-Wilcoxon test (**, P < 0.01). F. Average area of invasion of HCT116 cells transduced with empty vector (pLKO.1) or USP54-specific shRNAs (shUSP54.854 and shUSP54.856). Mann Whitney-Wilcoxon test was used to analyze statistical significance (*, P < 0.05; **, P < 0.01). G. Tumor xenograft model performed with subcutaneously injected control and USP54-depleted HCT116 cells. Data are presented as mean ± SEM and statistical significance was assessed by using a non-parametric Mann Whitney-Wilcoxon test (*, P < 0.05).

Figure 2. Usp54 is dispensable for embryonic development and adult mice lifespan

A., B. Kaplan-Meier survival curves for wild-type and Usp54-deficient males (A) and females (B). C. TaqMan-based qRT-PCR analysis of Usp54 in MEFs and liver tissues from Usp54^+/+ and Usp54^KF/KF mice. Data are represented as relative quantification, RQ ± SEM, two-tailed Student's t-test (**, P < 0.01). D. Body weight curves of Usp54^+/+ and Usp54^KF/KF female mice kept on standard diet. E. Body weight curves of Usp54^+/+ and Usp54^KF/KF female mice kept on high-fat diet and a representative image of females of each genotype at the end of the experiment. F. Total weight gain in the same animals. G. Percentage of gonadal and subscapular fat mass with respect to total body weight of the same animals. H. Mean adipocyte area in gonadal and skin fat. I. Representative histological images. Scale bar: 20 μm (gonadal fat) and 200 μm (skin fat). J. Average thickness of the subcutaneous fat deposits for each genotype. Statistical significance was assessed by a non-parametric Mann Whitney-Wilcoxon test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 3. USP54 deficiency prevents azoxymethane-induced infiltrating colon tumors

A. Schematic representation of azoxymethane-induced colon tumor protocol. B. Percentage of animals with the indicated histological alterations. L-Dys, light dysplasia; S-Dys, severe dysplasia; Adeno, adenocarcinomas; Inf. Adeno., infiltrating adenocarcinomas. C. Percentage of infiltrating and mucosal tumors within all carcinomas of each genotype and representative histological images. Scale bar: 500 μm. D. Average number of adenocarcinomas per mouse. E. Length of the colon at the end of the experiment. F. Analysis of USP54 expression from 32 colorectal cancer patient samples, comprising pairs of tumor and matched normal mucosa (GEO accession GDS2947). G. Kaplan-Meier survival plot for 269 patients with intestinal cancer grouped as a function of quantile expressions of USP54. Statistical significance was assessed by a non-parametric Mann Whitney-Wilcoxon test (*, P < 0.05; **, P < 0.01).

Figure 4. USP54 deficiency inhibits lung metastasis formation

A. TaqMan-based qRT-PCR analysis of Usp54 expression in B16F10 cells transduced with the indicated Usp54-specific shRNA or the empty lentiviral vector (pLKO.1) as a control. Data are represented as relative quantification, RQ ± SEM, two-tailed Student's t-test (***, P < 0.001). B. Number of metastases bigger than 200 μm in diameter. Statistical significance was assessed using a non-parametric Mann Whitney-Wilcoxon test (*, P < 0.05; ***, P < 0.001). C. Representative images of lungs and histological analysis for each condition. Scale bar: 200 μm.

Figure 5. USP54 alterations in human cancer

Summary of USP54 genetic alterations found in human cancer. ACC, Adrenocortical Carcinoma; Adeno, Adenocarcinoma; CCLE, Cancer Cell Line Encyclopedia; chRCC, Kidney Chromophobe; CS, Carcinosarcoma; DESM, Desmoplastic Melanoma; MPNST, Malignant Peripheral Nerve Sheath Tumor; NEPC, Neuroendocrine Prostate Cancer; PAAC, Acinar Cell Carcinoma of the Pancreas; SC, Small Cell; Sq and Squ, Squamous Cell Carcinoma; ucs, Uterine Carcinosarcoma. Data were obtained from cBioportal (http://cbioportal.org).