Inflammation suppresses DLG2 expression decreasing inflammasome formation

Abstract

Purpose: Loss of expression of DLG2 has been identified in a number of cancers to contribute to the disease by resulting in increased tumor cell proliferation and poor survival. In light of the previous evidence that DLG2 alters the cell cycle and affects proliferation, combined with indications that DLG2 is involved in NLRP3 inflammasome axis we speculated that DLG2 has an immune function. So far, there is no data that clearly elucidates this role, and this study was designed to investigate DLG2 in inflammatory colon disease and in colon cancer as well as its impact on inflammasome induction.

Methods: The DLG2 expression levels were established in publicly available inflammation, colon cancer and mouse model datasets. The overexpression and silencing of DLG2 in colon cancer cells were used to determine the effect of DLG2 expression on the activation of the inflammasome and subsequent cytokine release.

Results: The expression of DLG2 is repressed in inflammatory colon diseases IBD and Ulcerative colitis as well as colorectal cancer tissue compared to healthy individuals. We subsequently show that induction with inflammatory agents in cell and animal models results in a biphasic alteration of DLG2 with an initial increase followed by an ensuing decrease. DLG2 overexpression leads to a significant increase in expression of IL1B, IκBζ and BAX, components that result in inflammasome formation. DLG2 silencing in THP1 cells resulted in increased release of IL-6 into the microenvironment which once used to treat bystander COLO205 cells resulted in an increase in STAT3 phosphorylation and an increase proliferating cells and more cells in the G2/M phase. Restoration of DLG2 to the colon resulted in reduced AKT and S6 signaling.

Conclusion: DLG2 expression is altered in response to inflammation in the gut as well as colon cancer, resulting in altered ability to form inflammasomes.

Trial registration: NCT03072641.

Keywords: Colon cancer; DLG2; Inflammasome; NFKBIZ; Ulcerative colitis.

Conflict of interest statement

The authors have no relevant financial or non-financial interests to disclose. The authors declare that they have no conflicts of interest.

© 2022. The Author(s).

Figures

Fig. 1

The alteration of DLG2, NLRP3 and NFKBIZ gene expression in colon inflammation and cancer. aDLG2, eNLRP3 and iNFKBIZ gene expression by sample type including; 8 Healthy patients, 15 IBD patients, 15 adenoma patients and 15 colon cancer patients (cohort GSE4183). bDLG2, fNLRP3, and jNFKBIZ expression in 20 healthy individuals compared to 206 with Ulcerative colitis (UC) (cohort GSE109142). cDLG2, gNLRP3 and kNFKBIZ expression in 73 patients UC patients with active disease compared to 23 patients with inactive disease (cohort GSE75214). dDLG2, hNLRP3 and lNFKBIZ expression of 24 case-controlled tumor mucosa samples (cohort GSE10950). The expression data are presented as median centered log2 fold change and plotted as Tukeys box and whisker plots showing IQR, line at the median, + at the mean with whiskers ± 1.5-fold of interquartile range. Data outside the whiskers are shown as outliers. The paired data are shown as an individual symbol with a connecting line. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 2

The response of DLG2, NLRP3 and NFKBIZ gene expression to inflammation in mouse, cell and fly models. aDLG2, eNLRP3 and hNFKBIZ gene expression in the colon tissue of 5 mice for each time point in response to DSS treatment at 0, 2-, 4- and 6-days post treatment (cohort GSE22307). bDLG2, fNLRP3, and iNFKBIZ expression in the colon tissue of 4 mice in response to T-cell transfer at 0, 2-, 4- and 6- weeks post transfer (cohort GSE27302). cDLG2, gNLRP3 and jNFKBIZ expression in THP1 cells in response to LPS treatment at 0, 12 and 24 h. d The expression of dmDLG in Drosophila melanogaster gut in response to lactate bacteria treatment at 0, 1-, 2-, 3-, 4- days post treatment. The expression data are presented as median centered log2 fold change and plotted as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

The response of NFκβ members to increased DLG2 expression and activation of the inflammasome in THP-1 cells. The expression of aRELA, bRELB, cNFKB1 and dNFKBIZ in response to control conditions (circle symbol), LPS priming (square symbol) and LPS + ATP treatment (triangle symbol) with or without DLG2 overexpression. e Representative immunoblot showing the expression of; DLG2, p-P65 (Ser 536), RELA, RELB, p-P105 (Ser 932), NFκB1, IκBζ and GAPDH. Each experiment was performed in triplicate. The gene expression data are presented as log2 fold change and plotted as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

The response of cytokines and apoptotic proteins to increased DLG2 level and formation of the inflammasome in THP-1 cells. The gene expression of aIL1B, bIL6, cBAX and dBCL2 in response to control conditions (circle symbol), LPS priming (square symbol) and LPS + ATP treatment (triangle symbol) with or without DLG2 overexpression. e Representative immunoblot showing the expression of; BAX, BCL2, p-STAT3, STAT3, p-S6, S6, TIG1 and GAPDH. f Activation of the inflammasome in DLG2 silenced, control and DLG2 overexpressed THP1-ASC-GFP cells, in response to LPS and ATP treatment, as determined by PYCARD/ASC speck formation, presented as a percentage of observed cells. g The transfection efficiency of DLG2 in THP1 cells for silenced, control and DLG2 overexpression. Each experiment was performed in triplicate. The expression data are presented as log2 fold change and plotted as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5

The response of THP-1 cytokine production in colon cancer COLO205 cells. a Representative western blot showing the level of IL-1β and IL-6 production and release in the cell medium in DLG2 silenced, control and DLG2 overexpressed THP-1 cells in response to LPS and ATP, normalized to total protein level. b The effect of cell medium from DLG2 silenced (siDLG2), control and DLG2 overexpressed (DLG2) THP-1 cells on COLO205 cell proliferation, and c percentage of COLO205 cells in G2/M phase. Representative immunoblot showing the effect of THP-1 inflammasome growth media on the expression of d RELA, RELB, IκBζ, NFκB1, e BAX, BCL2, p-STAT3, STAT3, normalized to GAPDH. Each experiment was performed in triplicate. The expression data are presented as log2 fold change and plotted as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 6

The expression of DLG2 in colon cancer and the effect of low DLG2 level in colon cancer cells. a The expression of DLG2 in 20 paired samples from the healthy ascending colon (Healthy A), healthy descending colon (Healthy D), mucosa from colon cancer patients 10 cm adjacent to the tumor (Mucosa) and colon cancer tumor (Tumor). b TheDLG2 expression in colon adenomas comparing 32 samples < 1 cm, 16 samples between 1 and 1.5 cm and 15 samples larger than 1.5 cm. c The effect of DLG2 silencing (siDLG2) and overexpression (DLG2) on SW480 cell proliferation 48 h post transfection. d Kaplan–Meier survival curve showing the 5-year event free survival probability of colon cancer patients. e–g The gene expression of eNLRP3, fNFKBIZ, and gFOXO3 in response to DLG2 overexpression in SW480 cells. Normalized level of protein phosphorylation of h AKT1, i FOXO3 and j S6 in response to DLG2 overexpression in SW480 cells. k Representative immunoblot showing the effect of DLG2 overexpression on SW480 cells showing the expression of DLG2, p-AKT (s473), AKT1, p-FOXO3a (s318/321), FOXO3, p-S6 (s235/236) and S6, normalized to GAPDH. Each experiment was performed in triplicate. The expression data are presented as log2 fold change and plotted as either Tukey’s box and whisker plots showing IQR, line at the median, + at the mean with whiskers ± 1.5-fold of interquartile range or as mean. *p < 0.05, **p < 0.01, ***p < 0.001