Low NLRP3 expression predicts a better prognosis of colorectal cancer

Abstract

Background: NOD-like receptor pyrin domain-3 (NLRP3) inflammasome activation is a double-edged sword in tumorigenesis. Whether NLRP3 is involved in the progression and prognosis of colorectal cancer (CRC) remains elucidated and is the focus of the present study.

Methods: Immunohistochemistry (IHC) was applied on tissue microarray (TMA) to determine the expression of NLRP3 in CRC patients. All 100 patients were divided into the low NLRP3 group and the high NLRP3 group according to their NLRP3 IHC scoring. Additionally, CRC xenografts were established by injecting HCT116 or RKO cells subcutaneously into nude mice. Cell proliferation and apoptosis were determined in HCT116 cells after treatment with NLRP3 inhibitor MCC950.

Results: NLRP3 expression was up-regulated in colon adenocarcinoma tissues compared with that in paracancerous tissues in CRC patients, HCT116 xenograft, and RKO xenograft. High NLRP3 level correlated with the advanced TNM classification of malignant tumors, the occurrence of distant metastasis, vascular invasion, and positive lymph nodes. Furthermore, Kaplan-Meier survival analysis revealed that a high NLRP3 level was associated with a low 5-year survival rate and even a low 10-year survival rate. Moreover, the multivariable Cox proportional hazards regression model implied that NLRP3 expression level was an independent risk factor for CRC prognosis. Inhibition of NLRP3 by MCC950 suppressed cell proliferation, induced cell apoptosis, and decreased mRNA levels of interleukin 1β (IL1β) and interleukin 18 (IL18) in HCT116 cells.

Conclusions: High level of NLRP3 predicts poor survival in CRC patients. NLRP3 is a putative prognostic biomarker and a potential therapeutic target in CRC treatments.

Keywords: Survival analysis; colon adenocarcinoma; inflammasome; interleukin 18; interleukin 1β.

Figure 1. Up-regulation of NLRP3 in human colon adenocarcinoma tissues

(A) The H&E staining and IHC staining of NLRP3 expression in human colon adenocarcinoma tissues and paracancerous tissues, and quantification of IHC based on the histological score, n=60. (B) Western blot and quantification of NLRP3 in human colon adenocarcinoma tissues and paracancerous tissues, n=8/group. ^#P<0.05 vs. paracancerous tissues.

Figure 2. Up-regulation of NLRP3 in human colon adenocarcinoma xenografts

(A) H&E staining, IHC staining of NLRP3 in HCT116 xenografts and normal colons in nude mice, and quantification based on IOD. (B) Western blot and quantification of NLRP3 in HCT116 xenograft tissues and normal colons in nude mice. (C) H&E staining, IHC staining of NLRP3 in RKO xenografts and normal colons in nude mice, and quantification based on IOD. (D) Western blot and quantification of NLRP3 in RKO xenografts and normal colons in nude mice. n=6/group, *P<0.05 vs. normal colons.

Figure 3. High NLRP3 expression predicted poor survival in human colon adenocarcinoma

(A) High NLRP3 expression is associated with poor 5-year overall survival. (B) High NLRP3 expression is also associated with poor 10-year overall survival. n=33 and 67 in the low NLRP3 group and the high NLRP3 group, respectively.

Figure 4. Association between clinicopathological factors and 5-year overall survival in colon adenocarcinoma patients

Association between 5-year overall survival and age (A), infiltration degree (B), metastasis (C), positive lymph nodes (D), and TNM stage (E) in colon adenocarcinoma patients, respectively.

Figure 5. Independent risk factors for prognosis of patients with colon adenocarcinoma

The range of 95% confidence interval (CI) and P-value presents in the figure.

Figure 6. Inhibition of NLRP3 suppressed cell proliferation and induced cell apoptosis in HCT116 cells

HCT116 cells were seeded in six-well plates and treated with DMSO or MCC950 (2 μM) for 24 h. (A) Cell apoptosis was quantified by flow cytometric analysis of Annexin V-FITC and PI. (B) Cell cycle was quantified by flow cytometric analysis of PI. (C) Cell viability was determined by CCK-8. (D) The mRNA levels of IL1β and IL18 were quantified by qRT-PCR. n=3/group, ^#P<0.05 vs. DMSO group.