USP17 Suppresses Tumorigenesis and Tumor Growth through Deubiquitinating AEP

Abstract

Ubiquitin-specific protease 17 (USP17), a novel member of deubiquitinase, is reported to play essential roles in several solid tumors. However, the expression and function of USP17 in breast cancer tumorigenesis remains ambiguity. Here we found that the mRNA level of USP17 was lower in breast cancer tissues than normal tissues. Meanwhile, higher USP17 level was detected in normal epithelial cell MCF-10A and a less-malignant cell MCF-7 than malignant cell line MDA-MB-231. Inhibition of USP17 in MCF7 cells enhanced tumorigenesis and tumor growth while overexpression of USP17 in malignant MDA-MB-231 cells reduced its tumorigenesis and growth ability in vitro and in vivo. Further study revealed that USP17 interacted with and deubiquitinated Asparaginyl endopeptidase (AEP), resulting in decreased protein levels of AEP. Moreover, knockdown of AEP inhibited breast cancer tumorigenesis and growth in vitro and in vivo through the inactivation of ERK signaling. Taken together, our works indicate that USP17 deubiquitinates AEP, down-regulates its protein level, and inhibits breast cancer tumorigenesis through disturbing ERK signaling. Thus, our data suggests that USP17 is a potential tumor suppressor in breast cancer and AEP is a promising target in breast cancer therapy.

Keywords: AEP; Breast cancer; Deubiquitination; ERK; USP17.

Figure 1

USP17 expression in breast cancer cells and patients. A-B. Expression of USP17 in human breast cancer specimens using the GEPIA Database (A) and TCGA data from UALCAN(B). C. Expression of USP17 in different stages of breast cancer versus normal tissues from TCGA data. D. USP17 expression in a panel of breast cancer cell lines using CCLE database. E. qRT-PCR analysis of USP17 mRNA expression in MCF-10A, MCF-7 and MDA-MB-231 cell lines. F. Immunoblot analyses of USP17 in MCF-10A, MCF-7 and MDA-MB-231 cell lines.

Figure 2

Altered tumorigenesis and tumor growth of breast cancer cells by USP17 expression level. qRT-PCR analysis and Immunoblot analysis of USP17 expression in USP17 overexpressed MDA-MB-231 cells. qRT-PCR analysis and Immunoblot analysis of USP17 expression in USP17 knocked down MCF-7 cells. C-D. The cell growth curve of MDA-MB-231(C) and MCF-7 (D) cells with USP17 overexpression and USP17 knocked down. E-F. Tumor volume in mice injected with MDA-MB-231 cells (E) and MCF-7 (F) with USP17 overexpression and USP17 knocked down (n=5 for each group). All data are presented as the mean ± SD. *P < 0.05, **P <0.01.

Figure 3

USP17 deubiquitinates AEP. A. Co-IP analyses of AEP, USP17-Flag and TRAF6-Flag expression vectors in 293T cells and cell lysates with anti-AEP antibodies. B. Coelution of USP17, AEP and TRAF6 in lysates of 293T cells overexpressed USP17, AEP and TRAF6. C. Immunofluorescence staining of USP17 (red) and AEP (green) in 293T cells overexpressed USP17 and AEP. D. Immunoblot analysis of ubiquitination of AEP in MG-132 treatment 293T cells transfected with indicated plasmids.

Figure 4

USP17 down-regulates AEP protein level. A. Immunoblot analysis of AEP and Flag-tagged TRAF6, USP17 and USP17 C89S in MDA-MB-231 cell line. B. Immunoblot analysis of AEP in conditional medium of control and USP17 overexpressed MDA-MB-231 cells. C. Immunoblot analysis of AEP in MDA-MB-231 cells transfected with USP17 plasmids. D. qRT-PCR analysis of USP17 and AEP in MDA-MB-231 cells overexpressed USP17 and USP17C89S. E. qRT-PCR analysis of USP17 and AEP in USP17 knocked down MCF-7 cells.

Figure 5

AEP promotes breast cancer tumorigenesis. A. Immunoblot analysis of AEP in AEP knocked down MDA-MB-231 cells. B. The cell growth curve of MDA-MB-231 cells with AEP knocked down. C. MTT assay to detect cell number after AEP protein treatment in MDA-MB-231 cells. D. Cell cycle distribution in control and AEP knocked down MDA-MB-231 cells. E. Control and AEP knockdown MDA-MB-231 cells stably expressed GFP were injected into mammary fat pad of female nude mice. Representative tumors are shown by the fluorescent intensity. F. Quantification of the fluorescent intensity in E. G. Representative hematoxylin and eosin sections of tumors from E. H-I. Tumor volumes in mice injected with control, AEP knockdown, USP17 overexpression and AEP USP17 double overexpressed MDA-MB-231 cells.

Figure 6

USP17 inhibits AEP mediated activation of ERK signaling. A. Immunoblot analysis of p-ERK and ERK in MDA-MB-231 cells after treated with different concentration of AEP. B. Immunoblot analysis of p-ERK, ERK, p-p65 and p65 in control, USP17 overexpression and USP17 knockdown MDA-MB-231 cells.