TIPE Regulates DcR3 Expression and Function by Activating the PI3K/AKT Signaling Pathway in CRC

Abstract

Tumor necrosis factor-induced protein-8 (TIPE) is highly expressed in colorectal cancer (CRC). Decoy receptor 3 (DcR3) is a soluble secreted protein that can antagonize Fas ligand (FasL)-induced apoptosis and promote tumorigenesis. It remains unclear whether TIPE can regulate DcR3 expression. In this study, we examined this question by analyzing the relationship between these factors in CRC. Bioinformatics and tissue microarrays were used to determine the expression of TIPE and DcR3 and their correlation in CRC. The expression of TIPE and DcR3 in colon cancer cells was detected. Plasma samples were collected from CRC patients, and DcR3 secretion was measured. Then, dual-luciferase reporter gene analysis was performed to assess the interaction between TIPE and DcR3. We exogenously altered TIPE expression and analyzed its function and influence on DcR3 secretion. Lipopolysaccharide (LPS) was used to stimulate TIPE-overexpressing HCT116 cells, and alterations in signaling pathways were detected. Additionally, inhibitors were used to confirm molecular mechanisms. We found that TIPE and DcR3 were highly expressed in CRC patients and that their expression levels were positively correlated. DcR3 was highly expressed in the plasma of cancer patients. We confirmed that TIPE and DcR3 were highly expressed in HCT116 cells. TIPE overexpression enhanced the transcriptional activity of the DcR3 promoter. TIPE activated the PI3K/AKT signaling pathway to regulate the expression of DcR3, thereby promoting cell proliferation and migration and inhibiting apoptosis. In summary, TIPE and DcR3 are highly expressed in CRC, and both proteins are associated with poor prognosis. TIPE regulates DcR3 expression by activating the PI3K/AKT signaling pathway in CRC, thus promoting cell proliferation and migration and inhibiting apoptosis. These findings may have clinical significance and promise for applications in the treatment or prognostication of CRC.

Keywords: PI3K/AKT signaling pathway; apoptosis; cell proliferation; colorectal cancer; migration.

Figure 1

Tumor necrosis factor-induced protein-8 (TIPE) and DcR3 are highly expressed and positively correlated in colorectal cancer (CRC) patients. Box plots showing (A) TIPE and (B) DcR3 mRNA upregulation in CRC samples relative to normal samples (data downloaded from GEPIA). (C) The linear analysis relationship between TIPE and DcR3 showed that the proteins were positively correlated. (D) Relative DcR3 mRNA expression in UALCAN datasets, which included 41 normal samples and 274 samples with different stages of colorectal cancer. (E) Detection of relative TIPE mRNA expression in the same database. Kaplan-Meier curves for the overall survival of 177 CRC patients stratified by (F) TIPE and (G) DcR3 expression. *p < 0.05.

Figure 2

Tumor necrosis factor-induced protein-8 (TIPE) can upregulate DcR3 expression and positively regulate DcR3 transcription. (A) Comparison of TIPE and DcR3 mRNA expression in HCT116, and SW620 cells. TIPE and DcR3 mRNA expression was quantified by qRT-PCR and normalized in HCT116 cells. (B) Expression of TIPE and DcR3 in HCT116, and SW620 cells based on Western blot assays. (C) ELISA analysis and statistical analysis of DcR3 in a group of patients with CRC (n = 8) and healthy individuals (n = 4). (D) The relative luciferase activity of the DcR3 promoter was detected after TIPE overexpression and TIPE knockdown. (E) qRT-PCR assay of relative mRNA expression levels of DcR3 in two colon cancer cells based in TIPE overexpression or control cells. (F) DcR3 protein expression was detected by ELISA in supernatants of HCT116, and SW620 cells. TIPE promoted DcR3 secretion compared with that in the control group. ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

Alteration in tumor necrosis factor-induced protein-8 (TIPE) expression regulates cell proliferation and apoptosis in HCT116 cells. (A) TIPE was overexpressed or knocked down in HCT116 cells, and cell proliferation was measured by CCK-8 assays. (B) Cell proliferation assays of HCT116 cells transfected with TIPE and shTIPE after 24, 48, and 72 h of incubation. (C) HCT116 cells were transfected with TIPE or shTIPE for 24 (h) sFasL (100 ng/ml) was added for another 24 h, and the cells were fixed and stained with PI to analyze the DNA content with a CytoFlex S flow cytometer. The cell cycle phase (sub-G1, G1, S, and G2) is indicated. The sub-G1 phase is indicative of apoptosis. The experiment was performed three times with similar results. (D) Statistical analysis was used to assess the apoptosis rate of HCT116 cells after changes in TIPE,expression and sFasL treatment. ns, not significant, *p< 0.05, **p< 0.01, ***p< 0.001.

Figure 4

Changes in the expression of tumor necrosis factor-induced protein-8 (TIPE) affect cell migration. (A) After 12, 24, and 36 h incubation, in vitro wound healing assays with TIPE or shTIPE transfected HCT116 cells were performed to evaluate cell migration. (B) Histograms indicated the size of the wound healing area, and representative statistical results are shown. (C) Based on a Transwell assay, cell migration was significantly enhanced after transfection with TIPE in HCT116 cells but decreased after transfection with shTIPE. Quantitative analysis of three independent experiments is shown in (D); ns, not significant, **p< 0.01, ***p< 0.001.

Figure 5

Tumor necrosis factor-induced protein-8 (TIPE)-mediated activation of the PI3K/Akt pathway is involved in the modulation of DcR3 levels in HCT116 cells. (A) HCT116 cells were transfected with TIPE or an empty vector (control) for 24 h with lipopolysaccharide (LPS) stimulation for the indicated time (0, 15, 30, and 60 min). Cells were harvested, and whole-cell extracts were prepared for Western blot analysis of the indicated proteins. The blots shown are representative of those obtained in three separate experiments. (B) HCT116 cells were cultured in 6-well plates and transfected with TIPE or an empty vector (control) for 24 h, then qRT-PCR assays were performed to measure the relative DcR3 mRNA expression of HCT116 cells after treatment with LY294002 (50 μM), NSC23766, U0126, and BAY 11-7082. Expression was normalized to that in the control cells. (C) The culture medium was collected, and DcR3 protein levels were measured by ELISA. (D) HCT116 cells were transfected with tumor necrosis factor-induced protein-8 (TIPE) after treatment with lipopolysaccharide (LPS) for the indicated time (0, 30, and 60 min), and Western blot analysis of whole-cell lysates was performed to examine the indicated proteins in HCT116 cells after treatment with or without LY294002 (50 μM). ns, not significant, *p < 0.05, ***p < 0.001.

Figure 6

Tumor necrosis factor-induced protein-8 (TIPE) and DcR3 are upregulated in human colorectal cancer (CRC) and positively correlated with CRC metastasis. (A) Representative immunohistochemical detection of TIPE (left panel) or DcR3 (right panel) in tissue microarrays of CRC tissues and adjacent tissues. (B) Percentage of patients with high and low TIPE expression according to the following clinical parameters: tumor size (cm), tumor stage (I–II and III–IV), lymph node status (N0 or N1), and metastasis status (M0 or M1). (C) The percentage of patients with different expression levels of DcR3 according to different clinical parameters. ns, not significant, *p< 0.05.

Figure 7

Schematic diagram representing that TIPE regulates DcR3 expression and function by activating the PI3K/AKT signaling pathway in CRC. The solid line indicates direct regulation, the dashed line indicates indirect influence.