CTSG Suppresses Colorectal Cancer Progression through Negative Regulation of Akt/mTOR/Bcl2 Signaling Pathway

Abstract

Colorectal cancer (CRC) is the most common gastrointestinal tumor worldwide, which is a severe malignant disease that threatens mankind. Cathepsin G (CTSG) has been reported to be associated with tumorigenesis, whereas its role in CRC is still unclear. This investigation aims to determine the function of CTSG in CRC. Our results indicated that CTSG was inhibited in CRC tissues, and patients with CTSG low expression have poor overall survival. Functional experiments revealed that CTSG overexpression suppressed CRC cell progression in vitro and in vivo, whereas CTSG suppression supports CRC development cells in vitro and in vivo. Mechanistically, CTSG overexpression suppressed Akt/mTOR signaling mechanism and elevated apoptotic-associated markers, and CTSG silencing activated Akt/mTOR signaling mechanisms and inhibited apoptotic-associated markers. Furthermore, the Akt suppression signaling pathway by MK2206 abolishes CTSG-silenced expression-induced cell viability and Bcl2 up-regulation in vitro and in vivo. Altogether, these outcomes demonstrate that CTSG may act as a tumor suppressor gene via Akt/mTOR/Bcl2-mediated anti-apoptotic signaling inactivation, and CTSG represents a potential therapeutic target in CRC.

Keywords: Akt; Bcl2; CTSG; colorectal cancer; mTOR.

Figure 1

CTSG expression is down-regulated in human CRC tissues. (A-B) The data from TCGA showed that CTSG was significantly down-regulated in colorectal cancer tissues compared to the normal tissues (P < 0.001); (C) qPCR showed CTSG mRNA expression in 20 paired CRC and normal tissues (P < 0.05); (D) Western-blot analyzed the CTSG protein expression levels in 20 paired CRC and normal tissues; (E) Representative IHC staining of CTSG expression in CRC tissues and normal tissues; (F) Representative IHC staining of CTSG expression in TNM stage Ⅰ/Ⅱ and TNM stage Ⅲ/Ⅳ. Scale bar: 100 μm (100 ×) or 20 μm (400 ×). *P < 0.05, ***P < 0.001.

Figure 2

The correlations between clinicopathological features and CTSG expression. (A) Pie charts showing the Chi-squared test of clinicopathologic factors in high- and low-CTSG groups; (B) Patients in the high-CTSG group experienced a longer survival time tested by the Kaplan-Meier (KM) method; (C-D) Forest plots of univariate (C) and multivariate (D) Cox regression analyses in patients with CRC; (E) Nomogram model construction using CTSG and other clinical features; (F-H) 1- (F), 3- (G), and 5-year (H) AUC of the nomogram model, CTSG expression, and other clinical-pathological features were presented.

Figure 3

CTSG inhibits the proliferation of CRC cells in vitro. (A-D) Overexpression efficiency of HT29 (A) and HCT116 (B) cell lines, and knockdown efficiency in HCT116 (C) and RKO (D) cell lines; (E-F) Effects of CTSG overexpression on the proliferation of HT29 (E) and HCT116 (F) cells were monitored by MTT assays; (G-H) Effects of CTSG down-regulated expression on the proliferation of HCT116 (G) and RKO (H) cells were monitored by MTT assays; (I) Effects of CTSG overexpression in HT29 and HCT116 cells on colony formation; (J-K) Statistical results showed that the number of clones indicated in I; (L) Effects of CTSG down-regulated expression in HCT116 and RKO cells; (M-N) Statistical results showed the number of clones indicated in L. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

CTSG induces apoptosis of CRC cells in vitro. (A) Up-regulation or down-regulation of CTSG expression affected the apoptosis of colorectal cancer cells; (B-E) Statistical analysis indicated the difference in apoptosis rates in CRC cells, up-regulation of CTSG expression could promote CRC cell apoptosis (4B-C), whereas down-regulation of CTSG expression restrains CRC cell apoptosis (4D-E); (F-I) Apoptosis-related protein expression was examined by western-blot with CTSG overexpression in HT29 (F) and HCT116 (G) or with CTSG down-regulated expression in HCT116 (H) and RKO (I). **P < 0.01, ***P < 0.001.

Figure 5

CTSG mediates Akt/mTOR signaling pathway in CRC cell lines. (A) Volcano plot of DEGs in control and stable CTSG overexpression HT29 cells by RNA sequencing; (B) KEGG pathway analysis enriched Akt and mTOR signaling pathways; (C-D) Western-blot and histogram analysis of p-Akt and p-mTOR level in CTSG stable over-expression HT29 (C) and HCT116 (D) cell lines. (E-F) Western-blot and histogram analysis of p-Akt and p-mTOR level in CTSG stable knockdown expression HCT116 (E) and RKO (F) cell lines. *P < 0.05, **P < 0.01.

Figure 6

CTSG inhibits CRC cells proliferation in vivo. (A-E) CTSG overexpression inhibits proliferation: Photo of mice (A) injected with control and stable CTSG overexpression cells and tumor picture from mice (B), Tumor weight on the day the mice sacrificed (C), Tumor volume measured on every other day (D), and tumor volume on the day the mice sacrificed (E); (F-J) CTSG knockdown expression promotes proliferation: Photo of mice (F) injected with control and stable CTSG down-regulated expression cells and tumor picture from mice (G), Tumor weight on the day the mice sacrificed (H), Tumor volume measured on every other day (I), and tumor volume on the day the mice sacrificed (J); (K-L) Immunofluorescence staining for Ki67 in tumor tissues. Scale bar: 20 μm. *P < 0.05, **P < 0.01.

Figure 7

MK2206 suppresses CRC cell proliferation induced by CTSG silencing in vitro and in vivo. (A) Colony formation assay showed the effects of CTSG silencing cells treated with DMSO or MK2206 (5 μM) for ten days; (B) MTT assays showed the cell viability after treated with DMSO or MK2206 (5 μM); (C) The control cells and stable knockdown expression RKO cells were treated with DMSO or MK2206 (5 μM) for 24 h and then subjected to Western-blot analysis with the showed antibodies; (D-G) The control cells and stable knockdown expression RKO cells were injected subcutaneously into the flanks of mice, then treated with MK2206 (100 mg/kg) or equivalent volume of solvent. Mice and tumor pictures (D), tumor volume measured on every other day (E), tumor size (F), and weight (G) were recorded on the day the mice sacrificed. ns means no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 8

A proposed mechanism that CTSG suppresses the proliferation and promotes apoptosis of CRC cells. The figure is drawn by Figdraw (https://www.figdraw.com/).