Nucleotide-binding and leucine-rich repeat proteins (NLRPs), also known as NALPs, are crucial mediators in inflammation and host defense [1–3]. Currently, there are 10 known human NLRP genes (NLRP1-6, 9, 10, 12, 14). They, in common, possess pyrin, NACHT and leucine-rich repeat domains and are crucial for aggregating other proteins that form the inflammasome [1–3]. Of the 10 NLRP genes, gene functions of NLRP1, 3 and 5 have been known well [4–6]. NLRP3 inflammasome formation enables activation of caspase-1 and subsequent interleukin-1β and interleukin-18 activation, which could develop cell death and inflammation [7]. Both cell death and inflammation are crucial for cancer development, but the roles of NLRPs in cancer development remain controversial due to the diverse cancer-related findings [1, 5]. For example, recent studies identified NLRPs that modulated the mucosal immune response during inflammatory bowel disease-associated tumorigenesis [8, 9]. NLRP1 inflammasome attenuates colitis and colitis-associated tumorigenesis [9]. In skin squamous cell carcinoma, NLRP1 inflammasome pathway is silenced [10]. Also, NLRP2 inhibits cell proliferation and tumor growth of human glioblastoma [11]. NLRC3, a putative NLR, has anti-cancerous functions [12]. These data suggest tumor suppressor gene (TSG) functions of NLRPs in cancer development. By contrast, tumor-promoting (oncogenic) functions of NLRPs have been discovered in many cancers as well. NLRP1 overexpression is correlated with the tumorigenesis and proliferation of human breast cancer [13]. In melanoma, NLRP1 promotes tumor growth by enhancing inflammasome activation and suppressing apoptosis [14]. Activation of NLRP3 inflammasome promotes inflammation-induced tumor growth and metastasis in many cancers [2]. These data may indicate that NLRPs are involved in cancer pathogenesis, but their cancer-related alterations vary depending on cancer types.

DNA mismatch repair (MMR) is a cellular mechanism for correcting erroneous bases by MMR-specific proteins, alterations of which would result in microsatellite instability (MSI) and mutator phenotypes [15]. The mutator phenotype is characterized by mutation aCRCumulation in repetitive DNA sequences (frequently mono- or dinucleotide repeats). In coding DNA sequences, the MSI produces frameshift mutations within the affected proteins that would truncate protein synthesis [16]. Gastric (GC), colonic (CRC) and endometrial cancers are the most common cancers with high MSI (MSI-H) phenotype [17]. It is believed that MSI is random, but there is evidence suggesting that MSI targets include a growing list of cancer genes such as TGF-β1 gene and BAX gene [18, 19]. There are nucleotide repeats in coding DNA sequences of NLRP1, 2, 4 and 9, which could be altered in MSI cancers. In the present study, we detected frequent NLRP1, 2, 4 and 9 frameshift mutations in GC and CRC with MSI-H.

The role of inflammation is well-known in both development and progression of cancers, and is a hallmark in cancer development [23]. Also, evasion of cell death is known to play an important role in the pathogenesis of cancers [23]. In this sense, the inflammasome associated with both inflammation and cell death could be a good candidate for the cancer research. To find cancer-related alterations in cancer inflammasome, we analyzed frameshift mutations of NLRP (NLRP1, 2, 4 and 9) genes closely related to the inflammasome in GC and CRC in this study. The mutations were frequent in both MSI-H GC (15.5%) and CRC (5.5%); NLRP9 mutations revealed ITH; and NLRP9 expression was frequently lost in NLRP9-mutated cancers. These data indicate that NLRPs, especially NLRP9, might be involved in MSI-H cancer pathogenesis. In general, a cancer-related gene may exhibit not only a high incidence of genetic alterations but also functional consequences [23]. For NLRP9 gene, until now its role has been suggested only in maturation and fertilization of gonocytes [24]. Neither cancer-related functions nor gastrointestinal functions of NLRP9 gene have been identified. Mouse orthologue Nlrp9b is specifically expressed in intestinal epithelial cells and restricts rotavirus infection by activating pyroptosis [25]. It can be speculated that the NLRP9 frameshift mutations could inactivate the pyroptosis. However, relationship between pyroptosis and MSI cancers is not known currently. As The Human Protein Atlas database (https://www.proteinatlas.org/) exhibits that NLRP9 expression is evident in normal stomach and colon cells and is strongest in colorectal cancers followed by gastric, pancreatic, testicular and liver cancers, but other cancer tissues are in general weakly stained or negative, suggesting its role in gastrointestinal tract. Together, these data suggest that NLRP9 expression is common in both normal and cancer cells of stomach and colon, and that frameshift mutations and expression loss of NLRP9 is frequent in GC and CRC with MSI-H. However, it remains to be clarified whether these alterations have a causative role in tumorigenesis, or they simply reflect the phenomenon that MSI-H cancers have an increased frequency of mutations.

In addition to NLRP9, we found NLRP1, 2 and 4 mutations in GC and CRC with MSI-H with lower incidences (Table 2). As mentioned above [9, 13], NLRP1 possesses both oncogenic and TSG activities. Also, NLRP2 revealed TSG functions in glioblastoma and oncogenic functions in bladder cancer [26]. These opposing phenomenon may preclude predicting functional consequences of the frameshift mutations. NLRP4 negatively regulates autophagy processes through an association with beclin1 [27]. There is controversy about the roles of autophagy in cancer (tumor suppressing vs. promoting) [28]. The low incidence as well as the debate suggests that the NLRP4 mutation may not necessary play an important role in cancer development.

NLRP9 expression was negative in all cancers with its frameshift mutations (Table 3). Anti-NLRP9 antibody adopted in this study was made using a synthetic peptide of amino acids 472-549 (isoform 1). Based on the nucleotide changes of NLRP9 by the frameshift mutations, 2 amino acid locations (amino acids 7 and 264) would be altered and truncated (Table 2). Parts of pyrin domain (amino acids 1-94), NACHT domain (146-465) and leucine repeat domains (743-907) (www.uniprot.org) would be truncated by the mutations and thus the truncated area may not be detected by the immunohistochemistry.

Our data indicate that the mutational ITH of NLRP9 is common in the CRC (12.5% of MSI-H CRCs) (Figure 2). ITH is important in driving phenotypic selection and adaptation in response to selective pressures in a cancer [29]. ITH is known to be associated with poor prognosis and clinical outcomes [29]. The ITH in our study could ameliorate the phenotypic consequences of the mutation and further functional and clinical implication of the ITH is required to be defined.