Non-inflammasome forming NLRC3 suppresses tumorigenesis in the gut

Dysfunction of intracellular nucleotide-binding domain and leucine-rich repeats (NLRs, also known as NOD-like receptors) is associated with human diseases including infections, cancer, and autoimmune disease (1,2). These innate immune pattern recognition molecules (PRRs) are essential regulators of physiological and pathological inflammation (3,4). NLRs recognize pathogen- and danger-associated molecular patterns (PAMPs and DAMPs), before they mount immune responses to both microbial pathogens and damaged self, regulate tissue repair after damage and balance gut microbiota to maintain tissue homeostasis (5,6). Defective NLR-mediated signaling in the gut contributes to colitis and tumorigenesis of colorectal cancer (CRC) by increasing the permeability of the epithelial barrier, dysregulating the proliferation of epithelial cells, and inducing oncogenic mediators (3,7-9). All NLRs contain a central NACHT domain that facilitates oligomerization, a number of N-terminal effector domains that recruit downstream signaling molecules, and multiple C-terminal leucine-rich repeats (LRRs) for ligand sensing (3). Thus far, 23 human NLRs and NLR-like proteins can be distinguished by their effector domains that match the functional properties to each NLR (10). For example, the caspase activation and recruitment domain (CARD) distinguishes the NLRC subfamily (NLRC 1–5) and permits direct interaction between members of this family and other CARD carrying adaptor proteins (9). NLRC3 (also known as CLR16.2 or NOD3) is a poorly characterized member of the NLR family (11,12). Recent studies show that NLRC3 functions as a negative regulator of Toll-like receptor (TLR)-mediated signaling pathways (13) and the DNA sensor STING in response to PAMPs or to virus infection (14). Thus far, there are few reports on the role of NLRC3 in carcinogenesis. Published in Nature 2016, Dr. Karki and colleagues elegantly clarified the function of NLRC3 in colitis associated tumorigenesis (11).

With wild-type and Nlrc3^−/− mice, Karki and coauthors demonstrated a protective role of NLRC3 in tumorigenesis with two different murine models including the colitis-associated CRC model by treatment of azoxymethane and dextran sulfate sodium (AOM/DSS); and a spontaneous CRC model carrying a heterozygous mutation in the gene encoding adenomatous polyposis coli (Apc^Min/+). Compared to the wild-type mice, Nlrc3^−/− mice suffered significantly more high-grade dysplasia in the colitis-associated CRC model, where the extent or severity of damage was mostly appeared in the middle and distal colon and rectum (11). Furthermore, significantly increased inflammatory cytokines and chemokines in the colon tissue and systemically in the sera, as well as elevated levels of pro-inflammatory mediators such as IκBα of NF-κB pathways and STAT3 in the colon tissue were prominent in Nlrc3^−/− mice during disease transition from colitis to CRC. These data reflected the hyper-susceptibility of Nlrc3^−/− mice to colitis. Moreover, Apc^Min/+Nlrc3^−/− mice bore significantly higher percent of hyperplasia, exhibited increased damage in the colon where there were greatly increased number of Ki67+ proliferative cells compared to Apc^Min/+ mice as the control. Collectively, the Nature study from Dr Karki et al. on NLRC3 supplemented new evidence and strengthened, along with data from other NLRs, such as NOD1, NOD2, NLRP3, NLRP6, NLRC4, and NLRP12, the protective role of NLRs in tumorigenesis, mostly in colon cancer (3,7-10,15).

In Karki’s report, mice lacking NLRC3 especially in the intestinal epithelial cells developed the highest number of tumors, in comparison to mice lacking NLRC3 in hematopoietic cells, myeloid cells or the wild-type control (11). Further data showed NLRC3 inhibition on the hyper-­proliferation of enterocytes (11). In relevant studies, NLRs act in distinct cell types to prevent from tumorigenesis (15). For example, the expression of NLRC4 (16) or NLRP6 (17) in the gut, rather than hematopoietic cells, was essential to suppress the development of colon cancer; whereas sufficient expression of NLRP3 (18), or NLRP12 (19) in hematopoietic cells was necessary to attain the same function, although conflicting data exist (20). These data reflected the fact that, as innate immune sensors to protect and maintain intestinal integrity, these NLRs not only position in the gut but also reside systemically; In addition, Nlrc3^−/− mice had significantly higher levels of pro-inflammatory cytokines and chemokines than wild-type mice in the sera in the AOM/DSS model (11), indicating NLRC3 capability on regulating systemic inflammation that linked to CRC. Whereas it is possible that there may be a common pathway which NLRs converge on leading to protection against CRC, varied NLRs may well cooperate at different disease stages to maintain homeostasis in the gut where there is high host-microbiome interaction. Eventually, the activation of NLRs enables protective immune response against pathogens, or endogenous danger signals, while keeps tolerance to commensal microbes (1,5,15). Additional studies are warranted to elucidate these relevant mechanisms not only in animal models but also in relevant human diseases.

Previous research generally established two kinds of signaling cascades following the activation of NLRs and NLR-like proteins (9), both leading to inflammation. While NODs (including NOD1 and NOD2) form the Nodosome, other NLRs (i.e., NLRP1, NLRP3, NLRC4, etc.) assemble multi-protein inflammasome complexes of various platforms (8,15). Formation of Nodosome further activate canonical or non-canonical NF-κB signaling, as well as MAPK (p38, ERK, and JNK) pathways, leading to induction of anti-microbial peptides (AMPs), cytokines, and chemokines, and thereof inflammation. Inflammasome complexes allow cleavage and maturation of IL-1β and IL-18 and consequent inflammation and pyroptosis (6,8). In addition, activation of NF-κB, STAT3 or STAT1 signaling was involved in inflammasome-mediated inflammation (3). In Karki’s study, assessment of cytokine production of IL-1 and/or IL-18 in Nlrc3^−/− and wild-type mice revealed that NLRC3 (also known as NOD3) mediated inflammation in an inflammasome-independent manner (11). Moreover, the increased proliferation of intestinal epithelial cells and enhanced organoid formation in the absence of NLRC3 were highly related to activation of PI3K−Akt−mTOR pathways, which is commonly upregulated in human cancer (21); The treatment of a dual PI3K-mTOR inhibitor in Apc^Min/+Nlrc3^–/– mice greatly reduced the tumor burden to a similar level shown in control mice (11). Further analysis showed that NLRC3 inhibited PI3K activity via breaking up the association between its p85 with p110α subunit, and thereof downstream signaling events (11). Persistence of imbalanced nutrients and cellular energy, and cellular stress may well activate PI3K-mTOR signaling, the key regulator determining the cell fate via courses of cell apoptosis and/or autophagy, leading to a status of chronic inflammation that progressively results in cancer development (22). Importantly, the effective therapy with the inhibitor on PI3K-mTOR pathways in colitis-associated CRC in Karki’s study suggests future therapeutic directions to suppress the transition from chronic inflammation to cancer (11). Nonetheless, further studies are necessary to reveal specific ligands for NLRC3, and additional upstream signaling pathways in realizing NLRC3 functions during innate immune surveillance.

Compromised immune surveillance by chronic inflammation links to malignancy (6,22,23). Although a role of NLRC3 in the regulation of T-cell activation was previously indicated (24), the adaptive immunity including T and B cells seemed not involved in colitis-associated CRC in Nlrc3^−/− mice (11). Given that many NLR family members play a protective role in tumorigenesis (3,7-10,15), current data imply that there is strictly innate-mediated immune surveillance or tumor-suppressing mechanisms operated that inhibit cancer development without priming adaptive immune responses (4,25). These mechanisms need further investigations. NLRC3 was previously characterized as a negative regulator on inflammation and NF-κB pathways (10). Whereas inflammation is often necessary for eradication of pathogens and elimination of dangerous signals to eventually restore homeostasis, persistent NLR activation can cause significant collateral damage to the host tissue that eventually results in chronic inflammation and cancer (1,2,8). In summary, Karki and colleagues’ study expanded our understanding and elucidated new mechanisms on NLRC3, whose functions are mostly undiscovered, as compared to other NLRs (11). Their data may likely encourage an area of active investigations on the mechanisms and applications related to NLRs, innate immune surveillance, tumorigenesis and related therapeutics in human disease-associated situations, not solely on cancer.

Acknowledgments

Funding: This work is supported by the grant from Natural Science Foundation of China (NSFC-91229112).

Footnote

Provenance and Peer Review: This article was commissioned and reviewed by the Section Editor Zhikang Chen (Department of General Surgery, Xiangya Hospital, Central South University, Changsha, China) and Assistant Editor Dr. Chen Lai (Department of General Surgery, Xiangya Hospital, Central South University, Changsha, China).

Conflicts of Interest: Both authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/jxym.2017.07.08). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Kim YK, Shin JS, Nahm MH. NOD-like receptors in infection, immunity, and diseases. Yonsei Med J 2016;57:5-14. [Crossref] [PubMed]
2. Liu R, Truax AD, Chen L, et al. Expression profile of innate immune receptors, NLRs and AIM2, in human colorectal cancer: correlation with cancer stages and inflammasome components. Oncotarget 2015;6:33456. [Crossref] [PubMed]
3. Kent A, Blander JM. Nod-like receptors: key molecular switches in the conundrum of cancer. Front Immunol 2014;5:185. [Crossref] [PubMed]
4. Brubaker SW, Bonham KS, Zanoni I, et al. Innate immune pattern recognition: a cell biological perspective. Annu Rev Immunol 2015;33:257-90. [Crossref] [PubMed]
5. Jones JD, Vance RE, Dangl JL. Intracellular innate immune surveillance devices in plants and animals. Science 2016;354. [PubMed]
6. Ting JP, Willingham SB, Bergstralh DT. NLRs at the intersection of cell death and immunity. Nat Rev Immunol 2008;8:372-9. [Crossref] [PubMed]
7. Castaño-Rodríguez N, Kaakoush NO, Mitchell HM. Pattern-recognition receptors and gastric cancer. Front Immunol 2014;5:336. [PubMed]
8. Zhu H, Cao X. NLR members in inflammation-associated carcinogenesis. Cell Mol Immunol 2017;14:403-5. [Crossref] [PubMed]
9. Saxena M, Yeretssian G. NOD-Like Receptors: Master Regulators of Inflammation and Cancer. Front Immunol 2014;5:327. [Crossref] [PubMed]
10. Allen IC. Non-Inflammasome Forming NLRs in Inflammation and Tumorigenesis. Front Immunol 2014;5:169. [Crossref] [PubMed]
11. Karki R, Man SM, Malireddi RKS, et al. NLRC3 is an inhibitory sensor of PI3K–mTOR pathways in cancer. Nature 2016;540:583-7. [Crossref] [PubMed]
12. Karki R, Malireddi RS, Zhu Q, et al. NLRC3 regulates cellular proliferation and apoptosis to attenuate the development of colorectal cancer. Cell Cycle 2017;1243-51. [PubMed]
13. Schneider M, Zimmermann AG, Roberts RA, et al. The innate immune sensor NLRC3 attenuates Toll-like receptor signaling via modification of the signaling adaptor TRAF6 and transcription factor NF-κB. Nat Immunol 2012;13:823-31. [Crossref] [PubMed]
14. Zhang L, Mo J, Swanson KV, et al. NLRC3, a member of the NLR family of proteins, is a negative regulator of innate immune signaling induced by the DNA sensor STING. Immunity 2014;40:329-41. [Crossref] [PubMed]
15. Janowski AM, Kolb R, Zhang W, et al. Beneficial and Detrimental Roles of NLRs in Carcinogenesis. Front Immunol 2013;4:370. [Crossref] [PubMed]
16. Hu B, Elinav E, Flavell RA. Inflammasome-mediated suppression of inflammation-induced colorectal cancer progression is mediated by direct regulation of epithelial cell proliferation. Cell Cycle 2011;10:1936-9. [Crossref] [PubMed]
17. Normand S, Delanoye-Crespin A, Bressenot A, et al. Nod-like receptor pyrin domain-containing protein 6 (NLRP6) controls epithelial self-renewal and colorectal carcinogenesis upon injury. Proc Natl Acad Sci U S A 2011;108:9601-6. [Crossref] [PubMed]
18. Allen IC, TeKippe EM, Woodford RM, et al. The NLRP3 inflammasome functions as a negative regulator of tumorigenesis during colitis-associated cancer. J Exp Med 2010;207:1045-56. [Crossref] [PubMed]
19. Zaki MH, Vogel P, Malireddi RS, et al. The NOD-like receptor NLRP12 attenuates colon inflammation and tumorigenesis. Cancer Cell 2011;20:649-60. [Crossref] [PubMed]
20. Allen IC, Wilson JE, Schneider M, et al. NLRP12 suppresses colon inflammation and tumorigenesis through the negative regulation of noncanonical NF-κB signaling. Immunity 2012;36:742-54. [Crossref] [PubMed]
21. Shimobayashi M, Hall MN. Making new contacts: the mTOR network in metabolism and signalling crosstalk. Nat Rev Mol Cell Biol 2014;15:155-62. [Crossref] [PubMed]
22. Karin M, Lawrence T, Nizet V. Innate immunity gone awry: linking microbial infections to chronic inflammation and cancer. Cell 2006;124:823-35. [Crossref] [PubMed]
23. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011;144:646-74. [Crossref] [PubMed]
24. Conti BJ, Davis BK, Zhang J, et al. CATERPILLER 16.2 (CLR16. 2), a novel NBD/LRR family member that negatively regulates T cell function. J Biol Chem 2005;280:18375-85. [Crossref] [PubMed]
25. Teng MW, Swann JB, Koebel CM, et al. Immune-mediated dormancy: an equilibrium with cancer. J Leukoc Biol 2008;84:988-93. [Crossref] [PubMed]