Research report: NLRC4 Acts as a Non‐Canonical Tumour Suppressor in Lung Adenocarcinoma by Inhibiting PI3K/AKT/mTOR Signalling to Induce Protective Autophagy

The immune sensor NLRC4, long known for its role in triggering inflammatory cell death during bacterial infection, appears to lead a second, very different life inside lung cancer cells. A new study published in the Journal of Cellular and Molecular Medicine reports that NLRC4 is markedly depleted in lung adenocarcinoma, that low levels track with aggressive disease and shorter survival, and that restoring the protein suppresses malignant behaviour in cell and animal models. Strikingly, the mechanism has nothing to do with the inflammasome for which NLRC4 is famous. Instead, the researchers found that NLRC4 binds the transcription factor NFKB1, lifts repression of the tumour suppressor PTEN, and thereby calms the PI3K/AKT/mTOR signalling pathway, allowing a protective form of autophagy to switch on.

Lung cancer remains the most common cancer worldwide and the leading cause of cancer-related death. Non-small cell lung cancer accounts for roughly 85% of cases, and lung adenocarcinoma is its most prevalent subtype. Despite therapeutic advances, overall survival for lung adenocarcinoma patients remains unsatisfactory, which is why the molecular machinery driving progression continues to attract intense scrutiny. Autophagy, the cellular system that delivers cytoplasmic components to lysosomes for degradation, sits squarely in that machinery. It plays a double-edged role in cancer, suppressing early tumour formation while often being co-opted for survival in established malignancies, and the PI3K/AKT/mTOR axis is among the best-established and most frequently dysregulated regulators of the process.

To find new autophagy-related players in lung adenocarcinoma, the team, led by investigators at Shandong University, mined The Cancer Genome Atlas together with the Human Autophagy Database. From 30 differentially expressed autophagy-related genes, univariate Cox regression surfaced ten survival-linked candidates, and multivariate modelling distilled a six-gene prognostic signature comprising NLRC4, BIRC5, P4HB, ITGB4, DLC1 and ERO1A. NLRC4 carried the largest negative coefficient in the risk score, marking it as the signature’s predominant protective factor. Patients in the high-risk group of the model showed significantly poorer overall survival, and grade and risk score emerged as independent prognostic predictors in multivariate analysis.

The clinical case for NLRC4 grew stronger across independent datasets. In the TCGA lung adenocarcinoma cohort, NLRC4 transcripts were significantly downregulated in tumours, and high mRNA levels correlated with prolonged overall survival. The same favourable association held in four independent Gene Expression Omnibus validation cohorts: GSE50081, GSE31210, GSE11969 and GSE68465. Diminished NLRC4 expression also correlated with advanced clinical stage and lymph node metastasis in the public data, and subgroup analyses showed the prognostic value persisted regardless of EGFR or KRAS mutation status, suggesting the signal is independent of the major oncogenic drivers in this disease.

Crucially, the bioinformatics survived contact with real tissue. In an institutional cohort of 30 paired fresh-frozen lung adenocarcinoma tumours and matched adjacent non-tumour lung tissue from patients who had received no preoperative therapy, Western blotting showed marked downregulation of NLRC4 protein in tumours, with paired scatter analysis revealing a consistent decline across matched samples. PTEN, the lipid phosphatase that antagonises PI3K and serves as a master upstream gatekeeper of autophagy, fell in synchrony in the same patient lysates. Patients with low NLRC4 protein had significantly shorter overall survival, and low expression was significantly associated with advanced TNM stage and pleural invasion. Notably, the association with lymphatic metastasis did not reach significance in this small cohort, a reminder of its limits.

Enrichment analyses then pointed away from NLRC4’s day job. Gene Set Enrichment Analysis of the TCGA cohort showed autophagy gene sets prominently enriched in the NLRC4-high subgroup, KEGG analysis flagged the autophagy pathway, and Gene Ontology terms for autophagy initiation and intracellular catabolism were over-represented. NLRC4 expression correlated positively with core autophagy executors including BECN1, MAP1LC3B and ATG5. By contrast, its correlations with inflammasome and pyroptosis genes were weak or disjointed, including only a modest correlation with the essential adaptor PYCARD/ASC. High expression of the autophagy drivers ATG5 and BECN1 predicted favourable survival, mirroring NLRC4’s protective phenotype, whereas Gasdermin family pyroptosis executors conferred no such advantage.

Laboratory experiments converted correlation into mechanism. Across lung adenocarcinoma cell lines with distinct oncogenic backgrounds, including KRAS-mutant A549, EGFR-mutant H1975 and TP53-null H1299, NLRC4 overexpression raised Beclin-1, increased the LC3B-II/I ratio and promoted p62 degradation, while knockdown did the opposite. Transmission electron microscopy revealed denser autophagic vacuoles in overexpressing cells, and an mRFP-GFP-LC3B reporter showed a rise in red-only autolysosome puncta, indicating completed flux rather than stalled autophagosome accumulation. Blocking lysosomal degradation with bafilomycin A1 produced a larger net LC3B-II accumulation in NLRC4-overexpressing cells, confirming genuinely enhanced flux. Meanwhile, overexpression triggered no Caspase-1 or Gasdermin cleavage and no LDH release, even though LPS plus nigericin positive controls proved the pyroptotic machinery remained intact and responsive.

Functionally, NLRC4 behaved as a brake on malignancy, and autophagy was the brake cable. Overexpression impeded proliferation in CCK-8 assays, cut EdU incorporation, constrained wound closure and Matrigel invasion, and shifted EMT marker expression toward an epithelial profile with more E-cadherin and less N-cadherin, Vimentin and Snail. Chloroquine blockade of autophagy largely restored proliferation, migration and invasion in overexpressing cells, while rapamycin-induced autophagy rescued the aggressive phenotypes caused by NLRC4 knockdown. Silencing ATG5 genetically abolished the anti-proliferative effect of NLRC4 overexpression, sealing the dependency on intact autophagic machinery.

The upstream wiring proved equally instructive. NLRC4 overexpression lowered phosphorylated PI3K and AKT and total mTOR without changing total PI3K or AKT, and knockdown raised them. The AKT agonist SC79 reversed NLRC4-driven autophagy, while the PI3K inhibitor LY294002 restored flux in NLRC4-depleted cells, demonstrating that pathway suppression is functionally required. STRING database prediction and co-immunoprecipitation, both exogenous in HEK-293T cells and endogenous in lung cancer cells, confirmed a physical NLRC4-NFKB1 interaction that was markedly stronger in malignant A549 cells than in normal bronchial epithelial BEAS-2B cells. NLRC4 knockdown upregulated NF-kB target genes and accumulated phosphorylated p65 while abolishing PTEN protein, and NLRC4 and PTEN levels correlated positively across cBioPortal data and the 30-patient tissue cohort. Genetic rescue experiments then established PTEN as the indispensable mediator: PTEN knockdown blocked NLRC4-driven autophagy and tumour suppression, while PTEN restoration rescued the consequences of NLRC4 loss.

In vivo, the axis held. In female BALB/c nude mice bearing subcutaneous xenografts of PC9 cells, two independent NLRC4-targeting shRNAs accelerated tumour growth over four weeks and produced significantly heavier tumours than controls. Immunohistochemistry of the excised tumours showed reduced PTEN, elevated phosphorylated AKT and mTOR, more Ki-67-positive proliferating cells, accumulated p62 indicating impaired autophagic flux, and EMT marker shifts matching the in vitro data. The authors acknowledge limitations: direct NFKB1 occupancy at the PTEN promoter awaits chromatin immunoprecipitation and reporter assays, bulk transcriptomics may be confounded by stromal and immune cells, the xenografts were immunodeficient, and prospective cohorts will be needed to test NLRC4 as a stratification biomarker for PI3K pathway inhibitors. Even so, the study sketches a coherent, inflammasome-independent tumour-suppressive circuit in lung adenocarcinoma, and it suggests that measuring NLRC4 in biopsy specimens could eventually help identify patients whose tumours run on hyperactivated PI3K signalling.

Subject of Research: The non-canonical tumour-suppressive role of NLRC4 in lung adenocarcinoma via PTEN restoration and autophagy

Article Title: NLRC4 Acts as a Non‐Canonical Tumour Suppressor in Lung Adenocarcinoma by Inhibiting PI3K/AKT/mTOR Signalling to Induce Protective Autophagy

Article References: Zhang, Y., Liu, S., Li, R., Zhang, M., Yang, G., Zhu, X., & Qu, Y. (2026). NLRC 4 Acts as a Non‐Canonical Tumour Suppressor in Lung Adenocarcinoma by Inhibiting PI 3 K / AKT /m TOR Signalling to Induce Protective Autophagy. Journal of Cellular and Molecular Medicine, 30(20), Article e71420. https://doi.org/10.1111/jcmm.71420

Image Credits: AI Generated

DOI: 10.1111/jcmm.71420

Keywords: NLRC4, lung adenocarcinoma, autophagy, PTEN, PI3K/AKT/mTOR, NFKB1, pyroptosis, tumour suppressor, NF-kB, xenograft model, TCGA, inflammasome