Abnormally elevated USP37 expression in breast cancer stem cells regulates stemness, epithelial-mesenchymal transition and cisplatin sensitivity

Abstract

Background: Recent studies have indicated that deubiquitinating enzymes (DUBs) are related to the stem-cell pathway network and chemo-resistance in cancer. Ubiquitin-specific peptidase 37 (USP37), a novel DUB, was identified to be a potential factor associated with tumor progression. However, the biological functions of USP37 in breast cancer remain unclear.

Methods: The distribution of USP37 expression in breast cancer and the correlation between USP37 expression and the overall survival rate were detected by The Cancer Genome Atlas (TCGA) database. Gene set enrichment analysis (GSEA) was utilized to evaluate potential mechanism of USP37 in breast cancer. The USP37 expression in breast cancer tissues and breast cancer cell lines were detected by immunohistochemistry and western blotting. Sorting of breast cancer stem cells (BCSCs) were by using MACS assay. In vitro and in vivo assays were performed to examine the biological functions of USP37 in breast cancer cells. MG132, CHX chase, immunofluorescence staining and co-immunoprecipitation assays were used to test the interaction between USP37 and Gli-1.

Results: Bioinformatics analysis demonstrated that USP37 gene was elevated in breast cancer tissues and its overexpression was strongly correlated with the increased mortality rate. GSEA analysis showed that USP37 expression was positively associated with cell growth and metastasis while negatively related to cell apoptosis in the TCGA breast cancer samples. USP37 expression was elevated in breast cancer tissues and breast cancer cell lines. Moreover, we also detected that USP37 was overexpressed in BCSCs. USP37 regulated the ability of cell invasion, epithelial-mesenchymal transition (EMT), stemness and cisplatin sensitivity in breast cancer cell lines. Additionally, USP37 knockdown inhibited tumorigenicity and increased anticancer effect of cisplatin in vivo. Knockdown of USP37 significantly decreased hedgehog (Hh) pathway components Smo and Gli-1. Gli-1 was stabilized by USP37 and they interacted with each other. Further studies indicated that USP37 knockdown could inhibit the stemness, cell invasion and EMT in breast cancer via downregulation of Hh pathway.

Conclusions: These findings reveal that USP37 is highly expressed in BCSCs and is correlated with poor prognosis in breast cancer patients. USP37 can regulate the stemness, cell invasion and EMT via Hh pathway, and decreased USP37 confers sensitivity to cisplatin in breast cancer cells. USP37 is required for the regulation of breast cancer progression, as well as a critical target for clinical treatment of breast cancer.

Keywords: Breast cancer; Cisplatin; EMT; Hedgehog; Stemness; USP37.

Fig. 1

Overexpression of USP37 in human BC was correlated with poor-disease outcome. a Cancer with USP37 transcripts apparently had higher expression (n = 517) than normal breast tissues (n = 29) from the TCGA database [22] (***P < 0.0001). The median and interquartile range are represented by black lines. b USP37 transcripts among different subtypes are divided into five distinct molecular subtypes. The parameters were analyzed with the PAM50 gene expression profiling [22]. The median with interquartile range is represented by black lines. c Breast cancer (n = 738 patients) were evaluated with USP37 mRNA levels and results were correlated with overall survival over 25 years. The red line indicates patients with high USP37 transcript (n = 369) and blue line indicates patients with low USP37 transcript (n = 369). P value was analyzed by Kaplan-Meier analysis using GraphPad Prism. d-f GSEA analysis showed that USP37 expression was positively associated with metastasis (d) and cell growth (e) while negatively related to cell apoptosis (f) in the TCGA breast cancer samples. g The USP37 protein level in breast cancer tissues and surrounding tissues are shown by immunohistochemistry (IHC) (Brown: USP37). Scale bars: 100 μm. h USP37 IHC staining scores in breast cancer tissues (n = 60) and surrounding tissues (n = 60) are shown. **P < 0.01

Fig. 2

USP37 is highly expressed in breast cancer stem cells. a USP37 expression levels were detected in human normal breast epithelial cells (MCF-10A) and human breast cancer cells (MCF-7, MDA-MB-231, BT549 and T47D) via western blotting. *P < 0.05, **P < 0.01. b Protein expression levels of USP37 were tested in spheroid cells and adherent cells by western blotting. *P < 0.05, ***P < 0.001. c mRNA expression levels of USP37 confirmed in MCF-7 cell groups sorted by MACS by CD24 or CD44 marker by quantitative RT-PCR. ***P < 0.001. d Immunofluorescence staining of USP37 in BCSCs and non-BCSCs sorted by MACS with CD24 or CD44 marker in MCF-7 cells (Scale bar: 100 μm)

Fig. 3

Effect of USP37 expression on breast cancer cell EMT, migration and invasion. a MCF-7 and MDA-MB-231 cells were transfected with USP37 siRNAs or NC siRNA for 48 h. Western blotting analysis of USP37 knockdown efficiency in MCF-7 and MDA-MB-231 cells. Relative expression levels were analyzed by Image-Pro Plus 6.0 software. b Real-time quantitative PCR analysis of USP37 knockdown efficiency in MCF-7 and MDA-MB-231 cells. c, d USP37 knockdown significantly decreased N-cadherin, Snail1 and Vimentin as well as increased E-cadherin expression in MCF-7 and MDA-MB-231 cells. e Immunofluorescence staining of E-cadherin and N-cadherin after transfection with NC siRNA or USP37 siRNA#2 (Scale bar: 50 μm). f, g Representative images of migration assays for MCF-7 and MDA-MB-231 cells after downregulation of USP37 are shown at 12, 24 and 48 h (Scale bar: 200 μm). **P < 0.01, ***P < 0.001. h Representative images of Matrigel-based Transwell assay for MCF-7 and MDA-MB-231 cells after USP37 knockdown are shown at the 48 h time point (Scale bar: 200 μm).*P < 0.05. i MCF-7 cells were transfected with control plasmid (CTL) and a USP37 overexpression plasmid (USP37) for 48 h. USP37 upregulation significantly decreased E-cadherin and increased N-cadherin, Snail1 and Vimentin expressions in both MCF-7 and MDA-MB-231 cells. j Immunofluorescence staining of E-cadherin and N-cadherin after USP37 upregulation (Scale bar: 50 μm). k, l Representative images of migration and Matrigel-based Transwell assay for MCF-7 and MDA-MB-231 cells after USP37 upregulation (Scale bar: 200 μm).*P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

Downregulation of USP37 weakens cell self-renewal and chemo-resistance to cisplatin. a, b MCF-7 and MDA-MB-231 cells were transfected with shUSP37#2 and shScramble, and expressions of USP37, Smo, Gli-1, ALDH1 and OCT4 were confirmed by western blotting. *P < 0.05, **P < 0.01. c, d Comparison of mammosphere formation among inoculated cells in MCF-7 and MDA-MB-231 cells (original magnification, 4× or 20×).*P < 0.05, ***P < 0.001. e MCF-7-ShScramble and MCF-7-shUSP37#2 cells were treated with cisplatin (1 μg/ml) for 48 h, then treated cells were harvested to detect the levels of Bcl-2, Bax and cleaved caspase 9. GAPDH was examined as a loading control. j MDA-MB-231 cells transfected with shScramble or shUSP37#2 were exposed to 3 μg/ml cisplatin for 48 h, and the levels of Bcl-2, Bax and cleaved caspase 9 were detected via western blotting. f, g, k, l CCK-8 assay and colony formation assay showed that cell viability was decreased in MCF-7 and MDA-MB-231 cells after USP37 knockdown. Quantitation of colony formation in (h) MCF-7 and (i) MDA-MB-231. GAPDH was examined as a loading control. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

Overexpression USP37 elevates cell self-renewal and chemo-resistance to cisplatin. a Expressions of USP37, Smo, Gli-1, ALDH1 and OCT4 were detected in MCF-7-CTL or MCF-7-USP37 cells by western blotting. *P < 0.05, **P < 0.01. b Comparison of mammosphere formation among MCF-7-CTL or MCF-7-USP37 cells. *P < 0.05. c MCF-7-CTL and MCF-7-USP37 cells were treated with cisplatin (1 μg/ml) for 48 h, then treated cells were harvested in order to detect the levels of USP37, Bcl-2 and Bax. GAPDH was examined as a loading control. *P < 0.05. d CCK-8 assay showed that cell viability was enhanced in MCF-7 after USP37 upregulation compared to the control group. *P < 0.05, **P < 0.01, ***P < 0.01

Fig. 6

USP37 knockdown inhibits stemness, cell invasion and EMT via Hedgehog signaling pathway in breast cancer. a, b MCF-7 cells were incubated with 0.5 μM purmorphamine for 24 and 48 h. a Hedgehog pathway constituents were examined via western blotting. GAPDH was examined as a loading control. **P < 0.01,***P < 0.001. b Immunofluorescence staining images of MCF-7 cells showed the expression of USP37 and Hedgehog pathway constituents. c Protein levels of USP37, Smo, Gli-1, ALDH1, OCT4, E-cadherin, N-cadherin, Snail1 as detected by western blotting after the NC siRNA group or the USP37 siRNA#2 group was treated with 0.5 μM purmorphamine for 48 h. GAPDH was examined as a loading control. **P < 0.01,***P < 0.001. d Cell invasion capacity of the NC siRNA group or the USP37 siRNA#2 group treated with 0.5 μM purmorphamine (Scale bar: 200 μm). e Spheroid formation capacity of MCF-7-ShScramble or MCF-7-shUSP37#2 cells treated with 0.5 μM purmorphamine (original magnification, 4×). f Immunofluorescence staining of E-cadherin and N-cadherin after the NC siRNA group or the USP37 siRNA#2 group treated with 0.5 μM purmorphamine for 48 h. (Scale bar: 50 μm). *P < 0.05, **P < 0.01

Fig. 7

USP37 interacts with and stabilizes Gli-1 protein. a, b MCF-7 and MDA-MB-231 cells were transfected with shScramble and shUSP37#2, respectively, then treated with 10 μM of MG132 4 h before harvesting. c Immunofluorescence staining of Gli-1 after USP37 knockdown or USP37 overexpression (Scale bar: 50 μm). d, e Cells transfected with shScramble and shUSP37#2 were treated with 50 μg/ml of CHX and harvested at the indicated time point. f MCF-7-CTL and MCF-7-USP37 cells were incubated with 50 μg/ml of CHX and harvested at the indicated time point. Gli-1 expression was analyzed by western blotting and quantified with Image-Pro Plus 6.0 software. g Exogenous USP37 and Gli-1 magnified strong, positive overlapping expression in MCF-7 cells by immunofluorescent staining (Scale bar: 50 μm). h CO-IP assay showed the interaction between endogenous USP37 and Gli-1 in MCF-7 cells

Fig. 8

USP37 knockdown inhibits tumorigenicity and increases sensitivity to cisplatin in vivo. a, b, c MCF-7 cells transfected with shScramble or shUSP37#2 were subcutaneously injected into BALB/c nude mice treated with 0.9% saline or cisplatin. Tumors were excised from nude mice at day 28. a Images of tumor xenografts and b tumor weight for tumor xenografts treated with 0.9% saline or cisplatin are shown above. *P < 0.05, **P < 0.01. c Tumor growth curves were drawn every other day up to day 25. d Western blotting analysis of USP37, Smoothened, Gli-1, ALDH1, OCT4 and Bcl-2 in tumor xenografts. GAPDH was used as a loading control. *P < 0.05, **P < 0.01. e HE staining and immunohistochemical detection of USP37, Smoothened, Gli-1, Ki-67 in tumor xenografts. Scale bar: 100 μm