NLRP3 promotes tumor growth and metastasis in human oral squamous cell carcinoma

Inflammasomes are reported to be abnormally expressed and activated in several malignancies and play important roles in tumor development. The present study was designed to investigate the expression and function of the NLR family pyrin domain containing protein 3 (NLRP3) inflammasome in oral squamous cell carcinoma (OSCC).

Wang et al BMC Cancer (2018) 18:500 https://doi.org/10.1186/s12885-018-4403-9 RESEARCH ARTICLE Open Access NLRP3 promotes tumor growth and metastasis in human oral squamous cell carcinoma Han Wang1†, Qingqiong Luo1†, Xiaodong Feng1, Ruiyang Zhang1, Jiang Li2 and Fuxiang Chen1* Abstract Background: Inflammasomes are reported to be abnormally expressed and activated in several malignancies and play important roles in tumor development The present study was designed to investigate the expression and function of the NLR family pyrin domain containing protein (NLRP3) inflammasome in oral squamous cell carcinoma (OSCC) Methods: NLRP3 expression in OSCC cell lines and the normal human immortalized oral epithelial cells (HIOEC) was determined by real-time PCR and western blot Immunohistochemistry was used to examine the expression of NLRP3 and IL-1β in the paraffin-embedded OSCC tissues The proliferation of OSCC cells was detected by the 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and cell colony formation ability of the OSCC cells was also evaluated Tumor cell migration or invasion was measured by the transwell assay and related protein markers were determined by western blot A mouse xenograft model was established to investigate the OSCC tumor growth in vivo Results: Significant higher expression of NLRP3 was observed in the OSCC cells Obvious expression of NLRP3 and IL-1β was found in the paraffin-embedded OSCC tissues, and the NLRP3 expression levels were correlated with the tumor size, lymphonode metastatic status and IL-1β expression Downregulating NLRP3 expression markedly reduced the cleavage of caspase-1 and production of IL-1β in OSCC cells NLRP3 knockdown also inhibited the proliferation, migration and invasion of OSCC cells Further investigation indicated that expressions of E-cadherin and vimentin in OSCC cells were increased, while N-cadherin expression was decreased after NLRP3 knockdown Downregulating NLRP3 expression in OSCC cells significantly reduced the tumor growth in vivo Conclusions: Our data suggested that the increased expression of NLRP3 in OSCC was associated with tumor growth and metastasis NLRP3 may be considered as a potential target for OSCC therapy Keywords: NLRP3 inflammasome, Oral squamous cell carcinoma, Proliferation, Migration, Invasion Background Oral squamous cell carcinoma (OSCC) is the most common type of cancer in oral cavity, which accounts for more than 90% of all oral cancers [1] Approximately 300,000 new cases of OSCC patients occurs worldwide annually [2] Although there are much improvements in multimodality therapy including surgery, radiation and * Correspondence: chenfx@sjtu.edu.cn † Equal contributors Department of Clinical Immunology, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, 639 Zhizaoju Road, Shanghai 200011, China Full list of author information is available at the end of the article chemotherapy, the overall survival rate for OSCC still ranges from 50 to 60% [3, 4] Therefore, it is important to investigate the molecular mechanisms of the occurrence and development of OSCC, which may ultimately help the exploration and establishment of more effective therapeutic strategies Chronic inflammation has been reported to play critical roles in carcinogenesis and tumor progression [5–8] and cancer-related inflammation has been recognized as the “seventh hallmark of cancer” [9] Recently, inflammasomes-mediated inflammation in cancer has aroused great interests in the field [10, 11] © The Author(s) 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated Wang et al BMC Cancer (2018) 18:500 Inflammasomes are large protein complexes typically consisting of the nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR), adapter protein apoptosis-associated speck-like protein containing CARD (ASC) and caspase-1 [12, 13] Once activated by a diverse range of “danger signals” like oxidative stress, pathogens, metabolic and tissue damage products etc., the inflammasome complex subsequently cleaves pro-IL-1β to its mature bioactive form via the activated caspase-1 [14, 15] Studies of Fujita et al reported that the constitutively active NLR pyrin domain-containing protein (NLRP3) inflammasome in human melanoma cells mediated autoinflammation via caspase-1 processing and IL-1β secretion [16] and NLRP1 inflammasome activation promoted tumor growth in metastatic melanoma [17] Normand et al demonstrated the NLRP6 inflammasome controlled epithelial self-renewal and colorectal carcinogenesis upon injury [18] Kolb et al indicated obesity associated NLR family CARD domain-containing protein (NLRC4) inflammasome activation drove breast cancer progression [19] Other investigations have involved inflammasomes in bladder cancer [20], gastric cancer [21], and leukemia [22] However, the roles of inflammasomes, particularly NLRP3 inflammasome in OSCC have not been fully elucidated Thus we performed the present study and found that NLRP3 was aberrantly overexpressed in OSCC cells and tissues The NLRP3 expression in OSCC tissues was positively correlated with tumor size, lymphonode metastasis status and IL-1β expression (product of the activated NLRP3 inflammsome) Moreover, knockdown of NLRP3 inhibited OSCC cell proliferation, migration and invasion In the xenograft mouse model, we further demonstrated that suppressing NLRP3 expression could reduce the growth of OSCC in vivo Methods Patients and tissue specimens Seventy-seven primary OSCC tumor samples were collected from patients of Shanghai Ninth People’s Hospital at the Department of Oral and Maxillofacial–Head and Neck Oncology from January 2010 to July 2011, and all the patients had no previous chemotherapy or radiotherapy The pathological grades were evaluated according to the standard of American Joint Committee on Cancer (AJCC) and all study protocols were approved by the Ethics Committee of the hospital The clinical parameters of the OSCC patients were included in Table Animals Male BALB/c nude mice about four-week-old were purchased from Shanghai Laboratory Animal Center (Shanghai, China) and kept in animal care facilities under pathogen-free conditions The procedures of Page of 10 animal experiments were conducted in line with the institutional guidelines for the use of laboratory animals (The Ministry of Science and Technology of China, 2006) and also had approval of the Ethics Committee of the hospital Cell lines The human immortalized oral epithelial cells (HIOEC) and three OSCC cell lines- WSU-HN4, WSU-HN6 and CAL27 were kindly provided by the Key Laboratory of Stomatology, Shanghai Ninth People’s Hospital The OSCC cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (Invitrogen, Carlsbad, CA, USA) supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum (FBS) (Gibco, New York, NY, USA) HIOEC cells were cultured in defined Keratinocyte-SFM (Gibco) All cells were incubated in a humidified atmosphere containing 5% CO2 at 37 °C Immunohistochemistry (IHC) staining For IHC staining, the procedures were as described previously [23] After deparaffinating and rehydrating, the μm thick tumor tissue sections were heated by water bath with citric acid buffer for 20 and blocked in 5% normal goat serum for 30 min, and then incubated with either anti-NLRP3 antibody (1: 400, sigma, St Louis, MO, USA) or anti-IL-1β antibody (1:200, Abcam, Cambridge, UK) at °C overnight Subsequently, the GTVision Two-step Visualization System (Genetech, Shanghai, China) was used to visualize the immunostaining and hematoxylin was used to counter-stain Finally, the sections were examined under light microscopy and evaluated by the Immuno-Reactive-Score (IRS), which was calculated by multiplying staining proportion score (PS) and staining intensity score (IS) PS was defined as (0%), (1%~ 25%), (26%~ 50%), (51%~ 75%), and (76%~ 100%) IS was classified as (negative), (weak), (moderate) and (strong) According to the obtained IRS scores, patients were divided into three groups: negative expression group with IRS = 0, weak expression group with IRS = 1~ and strong positive expression group with IRS = 4~ 12 RNA extraction and real-time PCR To determine the mRNA expression of NLRP3, total RNA in HIOEC and OSCC cells was extracted using the TRIzol reagent (Invitrogen, San Diego, CA, USA) and real-time PCR were carried out as described previously [23] The PrimeScript RT Reagent kit (TaKaRa, Shiga, Japan) was adopted for reverse transcription Real-time PCR was carried out using the SYBR Premix Ex Taq II (TaKaRa) reaction system and cycling conditions were set according to the manufacturer’s instructions Wang et al BMC Cancer (2018) 18:500 Page of 10 Table Correlation between NLRP3 expression and clinicopathological features, IL-1β expression in patients with OSCC (n = 77) Characteristics n(77) P-valuea NLRP3 expression negative (20) weak(24) positive (33) n(%) n(%) n(%) Age(years) 0.436 ≤ 60 40 10 (25) 15 (37.5) 15 (37.5) >60 37 10 (27) (24.3) 18 (48.7) Male 57 15 (26.3) 19 (33.3) 23 (40.4) Female 20 (25) (25) 10 (50) Sex 0.718 Smoking statusb 0.491 Current/former 36 (22.2) 10 (27.8) 18 (50) Never 41 12 (29.3) 14 (34.1) 15 (36.6) Yes 26 (19.2) (30.8) 13 (50) No 51 15 (29.4) 16 (31.4) 20 (39.2) c Alcohol use 0.561 Location 0.403 Gingiva 23 (17.4) (26.1) 13 (56.5) Tongue 31 11 (35.5) 11 (35.5) (29) Floor of mouth 10 (10) (40) (50) Buccal 13 (30.7) (23.1) (46.2) I 21 10 (47.6) (38.1) 3(14.3) II 41 (19.5) 11(26.8) 22 (53.7) III 15 (13.3) (33.3) (53.4) T1 (55.6) (33.3) (11.1) T2 24 10 (43.5) (26.1) 8(30.4) T3 28 (10.7) 10(35.7) 15 (53.6) T4 16 (12.5) (31.2) 9(56.3) AJCC stage 0.018* T stage 0.015* N stage 0.008** N0 39 15(38.5) 15 (38.5) (23) N1 23 (13) (26.1) 14 (60.9) N2 15 (13.3) (20) 10 (66.7) IL-1β expression 0.004** Negative 21 12(57.2) 4(19) 5(23.8) Weak 21 4(19) 7(33.3) 10(47.7) Positive 35 4(11.4) 13(37.1) 18(51.5) P-values are based on chi-squared or Fisher’s exact test P < 0.05 indicates a significant association among the variables (*P