CSN6 Promotes the Migration and Invasion of Cervical Cancer Cells by Inhibiting Autophagic Degradation of Cathepsin L

Abstract

CSN6 is one subunit of the highly conserved constitutive photomorphogenesis 9 (COP9) signalosome (CSN), which is overexpressed in many types of cancers, and has received great attention as a regulator of the degradation of cancer-related proteins, suggesting its importance in oncogenic activity. CSN6 has been shown to be overexpressed in cervical cancer (CC) and associated with CC development. CC remains to be one of the most aggressive cancers affecting women. Cathepsin L (CTSL), significantly associated with the autophagy, plays a critical role in degradation of extracellular matrix for metastasis. However, the detailed biological functions of CSN6 on CTSL in CC metastasis have not been well clarified. Our data has shown that CSN6 and CTSL are positively correlated. The overexpression of CSN6 and CTSL might be a strong indicator for CC enhanced aggressiveness. CSN6 could suppress the degradation of CTSL, then facilitated the migration and invasion of CC cells. Interestingly, our results indicated that autophagy is essential for decreasing CTSL, while CSN6 could inhibit the autophagy ability of CC cells. In addition, blocking of the mammalian target of rapamycin (mTOR) pathway reversed CSN6-mediated autophagy inhibition. We further demonstrated that CSN6 positively regulated CTSL expression through an autophagy-lysosomal system. Taken together, we concluded that CSN6 might promote the migration and invasion of cervical cancer cells by inhibiting autophagic degradation of CTSL and serve as a potential gene therapy target for the treatment of CC metastasis.

Keywords: CSN6; CTSL; autophagy; cervical cancer; metastasis.

Figure 1

CSN6 and CTSL are both up-regulated in TMAs of CC, and correlated with 5-year overall survival in CC patients. (A) Representative photographs of immunohistochemistry staining for CSN6 and CTSL in human CC. Original magnification × 400 for A. (B) High expression of CSN6 was observed in 23.8% (10 of 42 cases) paracancerous tissues, while 56.3% (71 of 126 cases) CC tissues. (C) CTSL high expression staining was observed in 21.4% (9 of 42 cases) paracancerous tissues, and 53.2% (67 of 126 cases) CC tissues. (D) Kaplan-Meier survival analysis of the rate of overall survival according to low and high CSN6 expression of 126 patients with CC (P <0.001, Log rank test). (E) Kaplan-Meier survival analysis of 126 CC patients with high or low CTSL expression (P <0.001, Log rank test).

Figure 2

Effects of CSN6 on proliferation, motility, migration and invasion in CC cells. (A) Western blot assays were used to detect the expression of CSN6 in normal NCEC and CC cells (MS751, CaSki, HeLa, C33A, SiHa). β-actin served as an internal control. (B and C) Western blot analysis of CSN6 protein level after transfection of siCtrl, siCSN6,Vector and CSN6 expression plasmids in HeLa and SiHa cells. β-actin served as an internal control. (D and E) CCK-8 cell proliferation assays after CSN6 knockdown and overexpression for 1, 2 and 3 d in HeLa and SiHa cells. (F and G) Wound-healing assays were performed to examine the effect of CSN6 knockdown and overexpression on cell motility. Original magnification × 200 for F and G. (H and I) Transwell assays were utilized to determine the migration and invasion abilities after CSN6 knockdown and overexpression in HeLa and SiHa cells, respectively. Original magnification × 400 for H and I. All experiments were performed in triplicate. The data are presented as mean ± SD. **, P < 0.01; ***, P < 0.001.

Figure 3

Effects of CTSL on migration and invasion in CC cells. (A and B) Western blot assays were utilized to detect the expression of CTSL after transfection of siCtrl, siCTSL, Vector and CTSL expression plasmids in HeLa and SiHa cells. β-actin served as an internal control. (C and D) Transwell assays were performed to determine the migration and invasion abilities after CTSL knockdown and overexpression in HeLa and SiHa cells, respectively. Original magnification × 400 for C and D. All experiments were performed in triplicate. The data are presented as mean ± SD. ***, P < 0.001.

Figure 4

CSN6 promotes the cell migration and invasion by CTSL. (A) Western blot assays were used to detect the expression of CSN6 and CTSL in HeLa and SiHa cells, which were transfected of Vector and CSN6 overexpression plasmids before treating with or without CTSL inhibitor Z-Phe-Tyr-CHO (20 μM). β-actin served as an internal control. (B) The cell migration and invasion evaluated by transwell assays after three groups (Vector, CSN6, CSN6+Z-Phe-Tyr-CHO) in HeLa and SiHa cells. (C and E) Western blot analysis of CSN6 and CTSL expression in three groups of co-transfection as labeled. β-actin served as an internal control. (D) The cell migration and invasion evaluated by transwell assays after three groups of co-transfection (Vector+siCtrl, CSN6+siCtrl, CSN6+siCTSL) in HeLa and SiHa cells. (F) The cell migration and invasion evaluated by transwell assays after three groups of co-transfection (siCtrl+Vector, siCSN6+Vector, siCSN6+CTSL) in HeLa and SiHa cells. Original magnification ×400. All experiments were performed in triplicate. The data are presented as mean ± SD. **, P < 0.01; ***, P < 0.001.

Figure 5

Knockdown of CSN6 enhances autophagy in CC cells. (A) Western blot assays of CSN6, mTOR and LC3-I/II protein levels after CSN6 knockdown in HeLa and SiHa cells. β-actin served as an internal control. (B) Immunofluorescence staining of mTOR signal (green) in HeLa and SiHa cells was analyzed by fluorescent microscope after CSN6 knockdown. DAPI fluorescence signal occurred in the nucleus (blue). The intensity of mTOR per cell was quantified by densitometry (software: Image J, NIH). Original magnification ×400 for B. (C) The visualization of autophagic vacuoles in HeLa and SiHa cells was suggested by interspersed MDC labeling and analyzed by fluorescence microscopy. The number of MDC staining spots per cell was quantified. Original magnification ×400 for C. (D) HeLa and SiHa cells were first infected with adenovirus mRFP-GFP-LC3 (10 MOI) and further transfected with siCtrl and siCSN6 for another 48 hours. The number of yellow puncta and red only puncta per cell was quantified. Original magnification ×400 for D. (E) Western blot assays were used to detect the expression of CSN6 and LC3-II in HeLa and SiHa cells, which were transfected of siCSN6 before treating with or without lysosomal protease inhibitors E64d (10 μg/ml) and Pepstatin A (10 μg/ml). The integrated optical density (IOD) values of each bands were measured by Image J. The ratio of LC3-II/LC3-I was indicated graphically. All experiments were performed in triplicate. The data are presented as mean ± SD. ***, P < 0.001.

Figure 6

CSN6 inhibits autophagy by increasing mTOR expression in CC cells. (A) Western blot assays of LC3 and CTSL protein levels in HeLa and SiHa cells treated with mTOR inhibitor Rapamycin (RAPA: 0, 50, 100 nM) for 12 h. β-actin served as an internal control. (B) Western blot assays were used to detect the expression of CSN6, mTOR, LC3 and CTSL in HeLa and SiHa cells, which were transfected of Vector and CSN6 overexpression plasmids before treating with or without autophagy inducer RAPA(100 nM, 12 h). β-actin served as an internal control. (C) The cell autophagy flux of three groups (Vector, CSN6, CSN6+RAPA) in HeLa and SiHa cells pretreated with adenovirus RFP-GFP-LC3 (10 MOI). The number of yellow puncta and red only puncta per cell was quantified. Original magnification ×400 for C. All experiments were performed in triplicate. The data are presented as mean ± SD. **, P < 0.01.

Figure 7

CSN6 increases mTOR stability by reducing mTOR ubiquitination. (A) Western blot assays were used to detect the expression of CSN6 and mTOR in HeLa cells, which were transfected with Vector and CSN6 plasmids for 24 h and then treated with or without proteasome inhibitor MG132 (50 μg/ml) for 6 h before being harvested. (B) Western blot assays of mTOR turnover rate in CSN6 overexpressing HeLa cells. β-actin served as an internal control. HeLa cells were treated with cycloheximide (CHX) (100 μg /ml) for 0, 3, 6, 9 h after transfection. The IOD values of bands at each time point were measured by Image J. Remaining mTOR was indicated graphically. (C) HeLa cells were transfected with Vector or CSN6 plasmid. The cells were treated with MG132 (50 μg/ml) for 6 h before being harvested. The cell lysates were pulled down with anti-mTOR antibody and immunoblotted with anti-ubiquitin antibody. Western blot analysis of CSN6 and mTOR was performed using equal amounts of cell lysates. (D) HeLa cells were co-transfected with CSN6 and HA-ubiquitin plasmids. The cells were treated with MG132 (50 μg/ml) for 6 h before being harvested. The cell lysates were pulled down with anti-mTOR antibody and immunoblotted with anti-HA antibody. Western blot analysis of CSN6 and mTOR was performed using equal amounts of cell lysates. All experiments were performed in triplicate. The data are presented as mean ± SD. *, P < 0.05.

Figure 8

CSN6 interacts with CTSL and positively regulates CTSL expression through an autophagy-lysosomal system in HeLa cells. (A and B) Physical interaction of endogenous CSN6 with endogenous CTSL. Equal amounts of CSN6 overexpressing HeLa cells lysates were immunopricipitated with either IgG, anti-CSN6 or anti-CTSL antibody and immunoblotted with the indicated antibodies. Whole cell lysates (WCL) were immunoblotted with indicated antibody. (C and D) Western blot assays of CTSL turnover rate in CSN6 knockdown or overexpressing HeLa cells. β-actin served as an internal control. HeLa cells were treated with CHX (100 μg /ml) for 0, 3, 6, 9 h after transfection. The IOD values of bands at each time point were measured by Image J. Remaining CTSL was indicated graphically. (E and F) CSN6-mediated stabilization of CTSL was dependent on autophagy-lysosomal. CSN6 knockdown or overexpressing HeLa cells were treated with or without autophagy inhibitor Chloroquine (CQ, 10 μM) for 6 h before harvesting. Western blot analysis of CSN6 and CTSL protein levels. β-actin served as an internal control. All experiments were performed in triplicate. The data are presented as mean ± SD. *, P < 0.05.

Figure 9

CSN6 promotes the migration and invasion of HeLa cells through inhibiting autophagy. (A) Autophagy inducer RAPA was used to detect cell migration and invasion when overexpressing CSN6. The cell migration and invasion evaluated by transwell assays after three groups (Vector, CSN6, CSN6+RAPA) in HeLa cells. (B) Autophagy inhibitor CQ was used to detect the cell migration and invasion when knocking down CSN6 expression. The cell migration and invasion evaluated by transwell assays after three groups (siCtrl, siCSN6, siCSN6+CQ) in HeLa cells. Original magnification ×400 for A and B. All experiments were performed in triplicate. The data are presented as mean ± SD. ***, P < 0.001.

Figure 10

Model of CSN6 modulating CTSL stability and metastasis.