USP37 promotes angiogenesis and metastasis in colorectal cancer by facilitating β-catenin stability

Abstract

Ubiquitin-specific peptidase 37 (USP37) is a novel deubiquitinating enzyme, which has been found to be involved in the progression of multiple tumors. However, its function in colorectal cancer (CRC) remains unclear. Here, we firstly proved that USP37 was up-regulated in CRC cases, and high USP37 expression predicted poor survival of CRC cases. USP37 up-regulation promoted the proliferation, cell cycle progression, apoptosis inhibition, migration, invasion, epithelial mesenchymal transition (EMT) and stemness of CRC cells; moreover, USP37 facilitated the angiogenesis of human umbilical vein endothelial cells (HUVECs). However, USP37 silencing showed the opposite function. In vivo experiment suggested that USP37 silencing suppressed the growth and lung metastasis of CRC in nude mice. Interestingly, we found that CTNNB1 (gene coding for β-catenin) level was positively correlated with USP37 level in CRC and USP37 silencing suppressed the expression of β-catenin in CRC cells and xenograft tumor tissues. Further mechanistic studies showed that USP37 could enhance the stability of β-catenin by inhibiting its ubiquitination. Taken together, USP37 acts as an oncogene in CRC, which promotes angiogenesis, metastasis and stemness by enhancing β-catenin stability via inhibiting its ubiquitination. USP37 may be a usefully target in CRC clinical treatment.

Keywords: CRC; USP37; angiogenesis; metastasis; β-catenin stability.

Figure 1

USP37 expression was abnormally increased in CRC patients. A. USP37 level in COAD patients was analyzed according to the data collected from TCGA database. B. USP37 level in tumor tissues as well as paired adjacent normal tissues of 106 CRC cases was evaluated by qRT-PCR. C. The effect of USP37 level on the survival of 106 CRC cases was explored by Kaplan-Meier survival curve. D. IHC of clinical samples to monitor the expression of USP37 protein. Magnification: 100 × or 200 ×. E. Western blot of clinical samples to explore the level of USP37 protein. **P < 0.01 and ***P < 0.001.

Figure 2

USP37 intensified the proliferation and attenuated apoptosis of CRC cells. A. USP37 protein expression in normal human intestinal epithelial cell line (NCM460) and CRC cell lines (HCT116, SW620, LoVo, SW480 and T84) was researched by Western blot. B. Western blot was implemented to verify the transfection efficiency of CRC cells. C. MTT assay was employed to detect the proliferation capacity of CRC cells. D and E. Flow cytometry assay was executed to monitor the cell cycle and apoptosis of CRC cells. F. Cell cycle- and apoptosis-related protein expression was assessed by Western blot. **P < 0.01 vs. NCM460 cell line or Control group. ##P < 0.01 vs. Scramble group.

Figure 3

USP37 exacerbated the migration and invasion capacities of CRC cells. A. Wound healing assay was recruited to assess the migration capacity of CRC cells. Magnification: 200 ×. B. Transwell assay was executed to evaluate the invasion ability of CRC cells. Magnification: 200 ×. C. Sphere-forming assay was utilized to evaluate the stemness of CRC cells. Magnification: 200 ×. D. The expression of EMT- and stemness-related proteins was detected by Western blot. **P < 0.01 vs. Control group. ##P < 0.01 vs. Scramble group.

Figure 4

USP37 promoted the angiogenic ability of HUVECs. A. The culture medium of the transfected HCT116 and T84 cells was collected to culture HUVECs, and then the migration ability of HUVECs was evaluated by Transwell assay. Magnification: 200 ×. B. The culture medium of the transfected HCT116 and T84 cells was collected to culture HUVECs. Then angiogenesis assay was employed to explore the angiogenic ability of HUVECs. Magnification: 200 ×. C. The culture medium of the transfected HCT116 and T84 cells was collected to culture HUVECs. Then the VEGFA level in the culture medium of HUVECs cells was detected by ELISA. **P < 0.01 vs. Control group. ##P < 0.01 vs. Scramble group.

Figure 5

USP37 silencing attenuated the in vivo growth and lung metastasis of CRC. A. Photographs, volume and weight of xenograft tumor tissues were displayed. B. Immunohistochemistry of xenograft tumor tissues was performed to investigate the expression of Ki67 proteins. Magnification: 200 ×. C. Lung tissues of mice were experienced HE staining for the detection of nodules. Magnification: 200 ×. *P < 0.05 and **P < 0.01 vs. Scramble group.

Figure 6

USP37 expression was positively correlated with β-catenin in CRC. A. The correlation between USP37 and CTNNB1 (coding β-catenin protein) in CRC cases was analyzed based on the TCGA-COAD and TCGA-READ databases. B. USP37 and β-catenin expression in clinical CRC tissues was monitored by immunohistochemical. Magnification: 200 ×. C. USP37 and β-catenin expression detection in xenograft tumor tissues was executed by immunohistochemical. Magnification: 200 ×. **P < 0.01 vs. Scramble group.

Figure 7

USP37 could intensify the stability of β-catenin. A. The function of USP37 on the expression of β-catenin in CRC cells was investigated by Western blot. **P < 0.01 vs. Control group. ##P < 0.01 vs. Scramble group. B. Co-immunoprecipitation was performed on 293T cells to verify the interaction between USP37 and β-catenin. C and D. MG132 and CHX treatment was implemented on CRC cells to verify the mechanism of USP37 in regulating β-catenin expression.