Lih-Chyang Chen

Tumour inflammasome-derived IL-1β recruits neutrophils and improves local recurrence-free survival in EBV-induced nasopharyngeal carcinoma

Inflammasomes sense infection and cellular damage and are critical for triggering inflammation through IL‐1β production. In carcinogenesis, inflammasomes may have contradictory roles through facilitating antitumour immunity and inducing oncogenic factors. Their function in cancer remains poorly characterized. Here we show that the NLRP3, AIM2 and RIG‐I inflammasomes are overexpressed in Epstein‐Barr virus (EBV)‐associated nasopharyngeal carcinoma (NPC), and expression levels correlate with patient survival. In tumour cells, AIM2 and RIG‐I are required for IL‐1β induction by EBV genomic DNA and EBV‐encoded small RNAs, respectively, while NLRP3 responds to extracellular ATP and reactive oxygen species. Irradiation and chemotherapy can further activate AIM2 and NLRP3, respectively. In mice, tumour‐derived IL‐1β inhibits tumour growth and enhances survival through host responses. Mechanistically, IL‐1β‐mediated anti‐tumour effects depend on infiltrated immunostimulatory neutrophils. We show further that presence of tumour‐associated neutrophils is significantly associated with better survival in NPC patients. Thus, tumour inflammasomes play a key role in tumour control by recruiting neutrophils, and their expression levels are favourable prognostic markers and promising therapeutic targets in patients.

Heterogeneous Ribonucleoprotein K and Thymidine Phosphorylase Are Independent Prognostic and Therapeutic Markers for Nasopharyngeal Carcinoma

Purpose: Heterogeneous ribonucleoprotein K (hnRNP K) regulates thymidine phosphorylase (TP) mRNA stability. The aim of the present study was to analyze hnRNP K and TP expression in nasopharyngeal carcinoma (NPC) and to evaluate the prognostic and therapeutic potential of these two markers. Experimental Design: We analyzed hnRNP K and TP expression immunohistochemically in 121 clinically proven NPC cases. Statistical analyses were applied to correlate cytoplasmic hnRNP K with elevated TP expression and determine the prognostic significance of these parameters. The therapeutic implication of elevated TP expression was determined by measuring sensitivity of NPC cells to the TP-targeting drug, 5-fluoro-5′-deoxyuridine (5′-DFUR). Results: There was a high correlation between cytoplasmic hnRNP K and high TP (P < 0.001). Both cytoplasmic hnRNP K and high TP were associated with poor overall survival (OS; P = 0.007 and P < 0.001, respectively) and distant metastasis-free survival (P = 0.003 and 0.001, respectively) of NPC patients. A multivariate analysis confirmed that both cytoplasmic hnRNP K and high TP are independent prognostic predictors for OS (P = 0.020 and 0.010, respectively). NPC cells expressing high TP were more sensitive to treatment with the TP-targeting drug, 5′-DFUR. Conclusions: Cytoplasmic hnRNP K and high TP are associated with shorter OS and distant metastasis-free survival in NPC patients. In vitro experiments suggest that NPC tumors with high TP expression may be sensitive to 5′-DFUR treatment. Cytoplasmic hnRNP K and high TP may be potential prognostic and therapeutic markers for NPC, but additional validation studies are warranted.

A novel role for TNFAIP2: its correlation with invasion and metastasis in nasopharyngeal carcinoma.

Tumor necrosis factor alpha (TNFα) is an inflammatory cytokine that is present in the microenvironment of many tumors and is known to promote tumor progression. To examine how TNFα modulates the progression and metastasis of nasopharyngeal carcinoma, we used Affymetrix chips to identify TNFα-inducible genes that are dysregulated in this tumor. Elevated expression of TNFAIP2, which encodes TNFα-inducible protein 2 and not previously known to be associated with cancer, was found and confirmed by quantitative RT-PCR of TNFAIP2 expression in nasopharyngeal carcinoma and adjacent normal tissues. Immunohistochemical analysis showed that the TNFAIP2 protein was highly expressed in tumor cells. Analysis of 95 nasopharyngeal carcinoma biopsy specimens revealed that high TNFAIP2 expression was significantly correlated with high-level intratumoral microvessel density (P=0.005) and low distant metastasis-free survival (P=0.001). A multivariate analysis further confirmed that TNFAIP2 was an independent prognostic factor for nasopharyngeal carcinoma (P=0.002). In vitro, TNFα treatment of nasopharyngeal carcinoma HK1 cells was found to induce TNFAIP2 expression, and siRNA-based knockdown of TNFAIP2 dramatically reduced the migration and invasion of nasopharyngeal carcinoma HK1 cells. These results collectively suggest for the first time that TNFAIP2 is a cell migration-promoting protein and its expression predicts distant metastasis. Our data suggest that TNFAIP2 may serve as an independent prognostic indicator for nasopharyngeal carcinoma.

Aberrant methylation impairs low density lipoprotein receptor-related protein 1B tumor suppressor function in gastric cancer

DNA methylation plays a significant role in tumor progression. In this study, we used CpG microarray and differential methylation hybridization approaches to identify low density lipoprotein receptor‐related protein 1B (LRP1B) as a novel epigenetic target in gastric cancer. LRP1B was hypermethylated in four gastric cancer cell lines, and low LRP1B mRNA expression was associated with high methylation levels in gastric cancer cell lines. Addition of a DNA methylation inhibitor (5‐Aza‐dC) restored the mRNA expression of LRP1B in these cell lines, indicating that DNA methylation is involved in regulating LRP1B expression. In 45 out of 74 (61%) clinical samples, LRP1B was highly methylated; LRP1B mRNA expression was significantly lower in 15 out of 19 (79%, P < 0.001) gastric tumor tissues than in corresponding adjacent normal tissues. In addition, ectopic expression of mLRP1B4 in gastric cancer cell lines suppressed cell growth, colony formation and tumor formation in nude mice. These results collectively indicate that LRP1B is a functional tumor suppressor gene in gastric cancer and that is regulated by DNA methylation.

Pyk2 activates the NLRP3 inflammasome by directly phosphorylating ASC and contributes to inflammasome-dependent peritonitis

The inflammasome adaptor protein, ASC, contributes to both innate immune responses and inflammatory diseases via self-oligomerization, which leads to the activation of the protease, caspase-1. Here, we report that the cytosolic tyrosine kinases, FAK and Pyk2, are differentially involved in NLRP3 and AIM2 inflammasome activation. The inhibition of FAK and Pyk2 with RNA interference or chemical inhibitors dramatically abolished ASC oligomerization, caspase-1 activation, and IL-1β secretion in response to NLRP3 or AIM2 stimulation. Pyk2 is phosphorylated by the kinase Syk and relocalizes to the ASC specks upon NLRP3 inflammasome activation. Pyk2, but not FAK, could directly phosphorylate ASC at Tyr146, and only the phosphorylated ASC could participate in speck formation and trigger IL-1β secretion. Moreover, the clinical-trial-tested Pyk2/FAK dual inhibitor PF-562271 reduced monosodium urate-mediated peritonitis, a disease model used for studying the consequences of NLRP3 activation. Our results suggest that although Pyk2 and FAK are involved in inflammasome activation, only Pyk2 directly phosphorylates ASC and brings ASC into an oligomerization-competent state by allowing Tyr146 phosphorylation to participate ASC speck formation and subsequent NLRP3 inflammation.

Interactome-wide Analysis Identifies End-binding Protein 1 as a Crucial Component for the Speck-like Particle Formation of Activated Absence in Melanoma 2 (AIM2) Inflammasomes

Inflammasomes are cytoplasmic receptors that can recognize intracellular pathogens or danger signals and are critical for interleukin 1β production. Although several key components of inflammasome activation have been identified, there has not been a systematic analysis of the protein components found in the stimulated complex. In this study, we used the isobaric tags for relative and absolute quantification approach to systemically analyze the interactomes of the NLRP3, AIM2, and RIG-I inflammasomes in nasopharyngeal carcinoma cells treated with specific stimuli of these interactomes (H2O2, poly (dA:dT), and EBV noncoding RNA, respectively). We identified a number of proteins that appeared to be involved in the interactomes and also could be precipitated with anti-apoptosis-associated speck-like protein containing caspase activation and recruitment domain antibodies after stimulation. Among them, end binding protein 1 was an interacting component in all three interactomes. Silencing of end binding protein 1 expression by small interfering RNA inhibited the activation of the three inflammasomes, as indicated by reduced levels of interleukin 1β secretion. We confirmed that end binding protein 1 directly interacted with AIM2 and ASC in vitro and in vivo. Most importantly, fluorescence confocal microscopy showed that end binding protein 1 was required for formation of the speck-like particles that represent activation of the AIM2 inflammasome. In nasopharyngeal carcinoma tissues, immunohistochemical staining showed that end binding protein 1 expression was elevated and significantly correlated with AIM2 and ASC expression in nasopharyngeal carcinoma tumor cells. In sum, we profiled the interactome components of three inflammasomes and show for the first time that end binding protein 1 is crucial for the speck-like particle formation that represents activated inflammasomes.

A Novel Role of RASSF9 in Maintaining Epidermal Homeostasis

The physiological role of RASSF9, a member of the Ras-association domain family (RASSF), is currently unclear. Here, we report a mouse line in which an Epstein-Barr virus Latent Membrane Protein 1 (LMP1) transgene insertion has created a 7.2-kb chromosomal deletion, which abolished RASSF9 gene expression. The RASSF9-null mice exhibited interesting phenotypes that resembled human ageing, including growth retardation, short lifespan, less subcutaneous adipose layer and alopecia. In the wild-type mice, RASSF9 is predominantly expressed in the epidermal keratinocytes of skin, as determined by quantitative reverse-transcription PCR, immunofluorescence and in situ hybridization. In contrast, RASSF9−/− mice presented a dramatic change in epithelial organization of skin with increased proliferation and aberrant differentiation as detected by bromodeoxyuridine incorporation assays and immunofluorescence analyses. Furthermore, characteristic functions of RASSF9−/− versus wild type (WT) mouse primary keratinocytes showed significant proliferation linked to a reduction of p21Cip1 expression under growth or early differentiation conditions. Additionally, in RASSF9−/− keratinocytes there was a drastic down-modulation of terminal differentiation markers, which could be rescued by infection with a recombinant adenovirus, Adv/HA-RASSF9. Our results indicate a novel and significant role of RASSF9 in epidermal homeostasis.

ASC contributes to metastasis of oral cavity squamous cell carcinoma

ASC (Apoptosis-associated Speck-like protein containing a CARD) acts as a platform protein in the inflammasome cascade of some cancer types. However, its potential involvement in OSCC (oral cavity squamous cell carcinoma) has not yet been determined. Here, we investigated the potential role of ASC in OSCC. RT-qPCR analysis of 20 paired tumor and adjacent normal tissue samples revealed that the mRNA levels of ASC, along with IL-1β, CASP1, and NLRP3 in ASC-associated NLRP3 inflammasome were significantly elevated in OSCC tissues. Immunohistochemical staining of these four proteins in 111 clinical specimens revealed that high-level expression of ASC was significantly associated with tumor stage, node stage (p=0.001), overall stage (p<0.001), extracapsular spread (p<0.001), perineural invasion (p=0.004) and tumor depth (p<0.001). Kaplan-Meier survival analysis further revealed that high-level ASC expression was correlated with poorer overall survival (p=0.001), disease-specific survival (p<0.001) and disease-free survival (p<0.001). Studies using OSCC cell lines indicated that high-level ASC expression enhanced cell migration and invasion, and experiments using an orthotropic nude mouse model confirmed that ASC overexpression induced metastasis of OSCC cells. This is the first report to show that ASC contributes to OSCC metastasis, and that high-level ASC expression is a marker for poor prognosis in OSCC patients.