BackgroundProgrammed death-ligand 1 (PD-L1, CD274) is well known for its immunosuppressive function within the tumor microenvironment; however, its tumor cell-intrinsic roles remain incompletely characterized. Emerging evidence suggests that PD-L1 may regulate oncogenic processes beyond immune evasion. This study aimed to define the intrinsic functions of PD-L1 in non-small cell lung cancer (NSCLC), with a focus on autophagy and metastasis-related signaling pathways.MethodsIntegrated transcriptomic analyses of patient-derived NSCLC specimens were performed to evaluate associations between CD274 expression and oncogenic gene signatures. CRISPR-Cas9-mediated knockout and plasmid-driven overexpression of PD-L1 were conducted in H460 and A549 cell lines to assess proliferation, migration, clonogenicity, and 3D spheroid growth. Molecular interactions among PD-L1, TRAF6, and BECN1 were examined through immunoprecipitation and ubiquitination assays. Autophagy induction was evaluated by LC3 lipidation and autophagosome formation under Toll-like receptor (TLR) stimulation. The functional relevance of PD-L1 in metastasis was further assessed using xenograft models.ResultsClinical transcriptomic analyses demonstrated that CD274 upregulation correlates with enrichment of cancer progression, proliferation, and autophagy-associated gene sets in NSCLC. PD-L1 knockout markedly reduced cell proliferation, migration, clonogenicity, and 3D spheroid formation, whereas its overexpression enhanced these oncogenic phenotypes. Mechanistically, PD-L1 physically interacted with TRAF6 and BECN1, promoting TRAF6-dependent BECN1 ubiquitination and TLR-induced autophagy. PD-L1 depletion suppressed TLR-driven LC3 lipidation, autophagosome formation, and epithelial-mesenchymal transition (EMT), while PD-L1 overexpression augmented autophagy and EMT responses. In vivo, PD-L1-deficient lung cancer cells displayed diminished tumor growth and reduced metastatic potential in xenograft models.ConclusionsThis study identifies PD-L1 as a previously unrecognized intrinsic driver of NSCLC progression through activation of the TLR-TRAF6-BECN1 autophagy axis and promotion of EMT. Beyond its canonical role in immune evasion, PD-L1 functions as a dual-regulator of tumorigenesis by coordinating autophagy-dependent oncogenic processes. These findings provide novel mechanistic insight and support the therapeutic rationale for targeting PD-L1 not only as an immune checkpoint but also as a key modulator of cancer cell-intrinsic signaling in NSCLC.

Ubiquitin-specific proteases (USPs) are key regulators of protein homeostasis and have been implicated in various aspects of cancer development, including colorectal cancer (CRC). In this study, we investigated the role of USP11 in CRC pathogenesis. RNA-seq analysis of tumor and matched normal tissues from 35 CRC patients identified USP11 as significantly overexpressed in tumor samples. Elevated USP11 expression was correlated with reduced patient survival, suggesting its prognostic significance. Functional experiments using USP11-knockout and USP11-overexpressing CRC cell lines (HCT-15 and HT-29) revealed that USP11 promotes tumor cell proliferation, migration, colony formation, and 3D spheroid growth. Biochemical assays demonstrated that USP11 stabilizes EGFR and TRAF6 by removing K48-linked ubiquitin chains, thereby preventing their proteasomal degradation. These interactions potentiate both EGFR and Toll-like receptor (TLR) signaling pathways, contributing to CRC tumorigenesis. Loss of USP11 led to significant reductions in EGFR and TRAF6 protein levels, resulting in impaired tumorigenic behavior in vitro and in mouse xenograft models. Furthermore, USP11 deficiency suppressed tumor spheroid formation in response to EGF, HKLM (a TLR2 agonist), and LPS (a TLR4 agonist), whereas USP11 overexpression amplified these effects. Importantly, pharmacological inhibition of USP11 with mitoxantrone markedly decreased spheroid growth in both EGFR- and TLR-driven models, supporting its therapeutic potential. Overall, our findings reveal that USP11 contributes to CRC progression by stabilizing EGFR and TRAF6, thereby enhancing oncogenic signaling. These insights identify USP11 as a promising molecular target for CRC treatment and support the repurposing of mitoxantrone as an inhibitor of USP11-driven tumor growth.

Thioredoxin-interacting protein (TXNIP) functions as a tumor suppressor, but its role in lung cancer remains poorly defined. This study identifies TXNIP as a negative regulator of TNF receptor-associated factor 6 (TRAF6)-mediated NF-κB activation and autophagy, key pathways in tumor progression. TXNIP directly binds TRAF6 via its C-terminal arrestin domain, inhibiting TRAF6 dimerization and auto-ubiquitination. This, in turn, reduces ubiquitination of downstream targets TGF-β-activated kinase 1 and beclin 1 (BECN1), thereby suppressing NF-κB signaling and autophagic activity. TXNIP expression is significantly reduced in lung adenocarcinoma and lung squamous cell carcinoma, as demonstrated by public datasets and patient tissue analysis. Gene set enrichment analysis shows that non-small cell lung cancer patients with TXNIPDOWN and TRAF6UP expression exhibit increased metastasis-associated gene signatures and poorer survival outcomes. Functionally, TXNIP-knockout lung cancer cells show enhanced TRAF6 and BECN1 ubiquitination, increased LC3 puncta, and elevated NF-κB activity and cytokine production after TLR3/4 stimulation. These cells also display increased proliferation, migration, invasion, and colony formation in vitro across multiple lung cancer cell lines (A549 and H1299). Collectively, this study highlights TXNIP as a critical suppressor of TRAF6-driven oncogenic pathways in lung cancer, suggesting that its downregulation contributes to disease progression through enhanced TLR-induced signaling.

Non-small cell lung cancer (NSCLC) is a highly aggressive malignancy frequently driven by oncogenic mutations in the epidermal growth factor receptor (EGFR). Although EGFR-tyrosine kinase inhibitors (EGFR-TKIs) have shown clinical efficacy, challenges such as limited response duration and intrinsic mechanisms—such as EGFR amplification—can affect therapeutic outcomes. This study investigates the role of the USP21–EGFR–Lyn axis in NSCLC progression, identifying USP21 as a key regulator of EGFR and Lyn stability. Gene Set Enrichment Analysis (GSEA) of NSCLC patient datasets revealed a strong correlation between USP21 overexpression and poor prognosis. Functional studies using USP21-knockout (USP21-KO) lung cancer cell lines demonstrated reduced proliferation, migration, colony formation, and tumor spheroid growth. Mechanistically, USP21 interacts with EGFR and Lyn, preventing their ubiquitination and degradation, thereby sustaining oncogenic signaling. In vivo, USP21 depletion significantly suppressed tumor growth in xenograft models. Additionally, pharmacological inhibition of USP21 with BAY-805 effectively reduced EGF-induced tumor spheroid formation, highlighting its therapeutic potential. Collectively, these findings position USP21 as a promising target for NSCLC treatment and offer a potential approach to complement existing EGFR-targeted therapies.

Lung cancer, particularly non-small-cell lung cancer (NSCLC), remains a leading cause of cancer-related mortality worldwide. Recent studies have implicated pyrroline-5-carboxylate reductase 1 (PYCR1), a key enzyme in proline biosynthesis, in cancer progression, yet its specific role in lung cancer remains unclear. Here we demonstrate that PYCR1 plays a critical role in NSCLC progression through its functional association with the epidermal growth factor receptor (EGFR) and Toll-like receptor (TLR) signaling pathways. An analysis of patient datasets revealed that PYCR1 is upregulated in NSCLC tissues, with the enrichment of cancer-associated pathways in PYCR1-upregulated patients. Functional studies in PYCR1-knockout (PYCR1-KO) lung cancer cells generated via CRISPR–Cas9 showed reduced cell proliferation, migration, colony formation and tumor spheroid growth both in vitro and in vivo. Mechanistically, PYCR1 stabilizes EGFR by forming a complex with EGFR and USP11, thereby enhancing EGFR deubiquitination and stability. In addition, PYCR1 promotes TLR signaling by interacting with key downstream molecules, including TRAF6, TAK1, ECSIT and TAB2, facilitating their ubiquitination and NF-κB activation. The loss of PYCR1 attenuates EGFR- and TLR-induced signaling cascades, resulting in reduced activation of AKT, TAK1 and NF-κB. Importantly, treatment with PYCR1-IN-1, a selective PYCR1 inhibitor, significantly suppressed EGFR- and TLR-induced tumor spheroid growth in multiple lung cancer cell lines, underscoring PYCR1’s potential as a therapeutic target. Collectively, our findings establish PYCR1 as a critical regulator of EGFR and TLR signaling pathways, driving lung cancer progression. Targeting PYCR1 with pharmacological inhibitors such as PYCR1-IN-1 offers a promising strategy for combating EGFR- and TLR-driven NSCLC progression. Lung cancer is a major cause of cancer deaths, with non-small-cell lung cancer being the most common type. Researchers have found that an enzyme called pyrroline-5-carboxylate reductase 1 (PYCR1) might play a role in cancer growth, but its exact function in lung cancer is unclear. This study explores PYCR1’s role in lung cancer. The researchers used data from 42 patients with non-small-cell lung cancer to study PYCR1 levels in cancer tissues. They also created lung cancer cells without PYCR1 using CRISPR–Cas9 to see how it affects cell growth and movement. They found that removing PYCR1 reduced cancer cell growth and spread. They also tested a chemical that blocks PYCR1 and found it could slow down tumor growth. The study concludes that PYCR1 is important for lung cancer progression and could be a target for new treatments. This summary was initially drafted using artificial intelligence, then revised and fact-checked by the author.

Objective:Mitomycin C (MMC) is frequently used to prevent postoperative fibrosis in tracheal stenosis, yet its precise cellular mechanisms remain inadequately understood. This study aimed to elucidate the cytotoxic and autophagic effects of MMC on normal human tracheal fibroblasts (hTF) and human bronchial/tracheal epithelial cells (hTEC) to better understand its potential role in fibrosis regulation. Methods:hTF and hTEC were exposed to MMC at concentrations of 0.01, 0.1, and 1 μg/mL for 24, 48, and 72 h. Cell proliferation, autophagy induction, and the expression of autophagy-related proteins were assessed using viability assays and Western blot analysis. Additionally, the effects of MMC on cell migration and fibroblast-to-myofibroblast transition were investigated. Results:MMC partially reduced hTEC proliferation without inducing autophagy. In contrast, MMC significantly suppressed hTF growth in a dose- and time-dependent manner while promoting autophagy. Western blot analysis revealed increased expression of LC3, ATG5, and Rab7 in MMC-treated hTF, along with reduced cyclin D1 levels. Furthermore, MMC attenuated TGFβ-induced αSMA expression in fibroblasts, suggesting an inhibitory effect on fibrosis-related cellular transformation. Conclusion:These findings indicate that MMC suppresses human tracheal fibroblast proliferation through autophagy-mediated cell death while sparing epithelial cells. This dual effect underscores its potential as a targeted antifibrotic agent for tracheal stenosis management. Further research is needed to optimize MMC's application and elucidate its long-term impact on airway remodeling. Level of Evidence:5.

The emerging role of ubiquitin-specific peptidase 21 (USP21) in stabilizing Fra-1 (FOSL1) highlights its involvement in promoting colorectal cancer (CRC) metastasis. Additionally, a reciprocal link between EGFR signaling and Fra-1 activation has been identified, mediated through matrix metalloproteinases (MMPs). However, the functional implications of the USP21-EGFR signaling axis in metastatic CRC (mCRC) are not fully understood. To investigate the clinical correlation between USP21 and EGFR expression, RNA-Seq data from tumor tissues (n = 27) and matched normal tissues (n = 27) of 27 mCRC patients were analyzed. Functional studies were performed, including the use of CRISPR/Cas9 to generate USP21-knockout (USP21-KO) CRC cells, in vitro assays for cancer progression and tumor formation, in vivo xenograft assays in NSG mice. Additionally, the therapeutic effect of the USP21 inhibitor, BAY-805, was evaluated. We found that elevated levels of USP21 and EGFR expression in mCRC patients were associated with poorer survival outcomes. Mechanistically, USP21 was found to enhance EGFR stability by deubiquitinating EGFR, leading to reduced EGFR degradation. USP21-KO colon cancer cells exhibited significantly reduced proliferation, migration, colony formation, and 3D tumor spheroid formation in response to EGF. Furthermore, the tumorigenic activity in vivo was markedly diminished in NSG mice xenografted with USP21-KO colon cancer cells. Importantly, BAY-805 demonstrated a notable inhibitory effect on the formation of 3D tumor spheroids in colorectal cancer cells stimulated with EGF. These findings suggest that USP21 could be a valuable therapeutic target and predictive biomarker for managing mCRC driven by EGF.

Protein tyrosine kinase 2 (PTK2), epidermal growth factor receptor (EGFR), and toll-like receptor (TLRs) are amplified in non-small cell lung cancer (NSCLC). However, the functional and clinical associations between them have not been elucidated yet in NSCLC. By using microarray data of non-small cell lung cancer (NSCLC) tumor tissues and matched normal tissues of 42 NSCLC patients, the genetic and clinical associations between PTK2, EGFR, and TLRs were analyzed in NSCLC patients. To verify the functional association, we generated PTK2-knockout (PTK2-KO) lung cancer cells by using CRISPR-Cas9 gene editing method, and performed in vitro cancer progression assay, including 3D tumor spheroid assay, and in vivo xenografted NSG (NOD/SCID/IL-2Rγnull) mouse assay. Finally, therapeutic effects targeted to PTK2 in lung cancer in response to EGF and TLR agonists were verified by using its inhibitor (Defactinib). In summary, we identified that up-regulated PTK2 might be a reliable marker for EGFR- or TLRs-induced lung cancer progression in NSCLC patients via the regulation of the cross-talk between EGFR- and TLRs-mediated signaling. This study provides a theoretical basis for the therapeutic intervention of PTK2 targeting EGFR- or TLRs-induced lung cancer progression.

Dear Editor, Chemokines and their receptors play essential roles in neoplastic transformation, tumour cell growth and survival, and organ-specific metastasis during carcinogenesism.1-3 Of various CXC chemokines, CXCL13 and its related receptor CXCR5 have been implicated in lung cancer progression.2 However, the molecular and cellular mechanisms by which the CXCR5–CXCL13 signal axis is functionally regulated in lung cancer progression are still poorly understood. Through clinical microarray data analysis of primary non-small cell lung cancer (NSCLC; n = 42) patients, we found that the up-regulation of CXCR5 and CXCL13 or CXCR5 and TLR4 in lung tumour tissues versus matched lung normal tissues was significantly associated with gene sets related to cancer module, lung fibrosis, VEGF, chemokine, cytokine and TLR signalling pathway. Through functional analysis with CXCR5-knockout (CXCR5-KO) human lung cancer cells generated by CRISPR/Cas9 gene editing method, we found that the CXCR5–CXCL13 axis was functionally linked to TLR4 signalling through activation of NF-κB for lung cancer progression, strongly suggesting that our clinically comparative results and functional investigations of TLR4–CXCR5 signalling network in lung cancer could potentially contribute to translational approaches for the development of lung cancer therapeutic agents. Gene expression profiling interactive analysis (http://gepia.cancer-pku.cn/detail.php?gene = CXCR5) revealed a positive correlation between CXCR5 and CXCL13 expression in lung adenocarcinoma (LUAD) (Figure S1A; p = 3.8e−08, R = 0.25). The expression of CXCL13 was significantly enhanced in LUAD and lung squamous cell carcinoma (Figures S1B and C). To clinically get insight into the role of the CXCR5–CXCL13 axis, we utilised microarray data of primary NSCLC patients’ lung tumour tissues (n = 42; Table S1) and their matched lung normal tissues (n = 42). We performed a gene set enrichment analysis (GSEA; https://www.gsea-msigdb.org/gsea/index.jsp) to identify biological processes and pathways associated with the expression of CXCR5 and CXCL13. By differential magnitudes (△Mag) of CXCR5 and CXCL13 expression between lung tumour tissues and matched lung normal tissues, we sorted and selected 21 LTTs for the GSEA (Figure 1A, 14 CXCR5upCXCL13up LTTs and seven CXCR5downCXCL13down LTTs; Table S2). GSEA results revealed that 13 cancer module gene sets were significantly enriched in CXCR5upCXCL13up LTTs versus CXCR5downCXCL13down LTTs (Figures 1B–G and S2A–G). Moreover, gene sets related to lung fibrosis, VEGF, chemokine signalling, cytokine and JAK–STAT signalling pathways were highly enriched in CXCR5upCXCL13up LTTs compared with those in CXCR5downCXCL13down LTTs (Figures 1H–L), suggesting that expression levels of CXCR5 and CXCL13 might be associated with lung cancer. To functionally verify the role of CXCR5, CXCR5-KO A549 and H1299 lung cancer cells were generated using CRISPR/Cas9 gene-editing method (Figures 1M and N, CXCR5-KO A549; Figure 1O, CXCR5-KO H1299).4, 5 Wound healing assay and transwell assay to evaluate cancer cell migration, cell proliferation assay and anchorage-dependent or -independent colony formation assay were performed using control (Ctrl) and CXCR5-KO lung cancer cells treated with or without CXCL13. Cancer cell migration was significantly induced in Ctrl A549 and Ctrl H1299 cells treated with CXCL13, whereas it was markedly attenuated in CXCR5-KO A549 and CXCR5-KO H1299 cells (Figures 2A–D, wound healing assay; Figures 2E–H, transwell assay). Upon CXCL13 stimulation, cell proliferation and anchorage-dependent colony formation ability were markedly attenuated in CXCR5-KO A549 or CXCR5-KO H1299 cells treated with CXCL13 as compared to those in Ctrl A549 or Ctrl H1299 cells treated with CXCL13 (Figures 2I–J, cell proliferation; Figures 2K–N, anchorage-dependent colony formation). Similar results were observed in anchorage-independent colony formation assay (Figures S3A–D). Taken together, these results suggest that the CXCR5–CXCL13 signalling axis is functionally implicated in lung cancer progression. Previous studies have shown that TLRs, such as TLR2 and TLR4, contribute to cell proliferation by modulating the CXCR5–CXCL13 signalling axis and regulate lung cancer progression.6-8 Importantly, CXCR5-knockdown cells exhibit attenuation of the activation of NF-κB induced by a TLR4 agonist LPS,9 suggesting that the CXCR5 signal might be functionally associated with the TLR4 signal through NF-κB activation. Interestingly, we found that gene sets related to innate responses, such as TLR signalling pathway, TNF-signalling via NF-κB, IL-6 pathway, local acute inflammatory response, cytokine–cytokine receptor interaction, CXCR3 pathway, AP1 pathway and chemokine receptors bind chemokines, were significantly enriched in CXCR5upCXCL13up LTTs versus CXCR5downCXCL13down LTTs (Figures 2O–R and S4A–D). To get insight into the association between TLR4 and CXCR5 in lung cancer, we further selected five CXCR5upTLR4up LTTs and 10 CXCR5downTLR4down LTTs in 42 NSCLCs (Figure 2S and Table S2) and performed GSEA. Ten gene sets related to cancer modules were highly enriched in five CXCR5upTLR4up LTTs versus 10 CXCR5downTLR4down LTTs (Figures 2T–X and S5A–E). Additionally, gene sets related to the TLR signalling pathway, cytosolic DNA sensing pathway, NOD-like receptor signalling pathway, RIG-I-like receptor signalling pathway, and complement cascade pathway were significantly enriched in five CXCR5upTLR4up LTTs (Figures 3A and S6A–D). To explore the functional effect between TLR4 and CXCR5 signals, we performed biochemical studies. Upon TLR4 stimulation with LPS, the expression of CXCR5 was significantly increased in A549 cells (Figure 3B, lane 2−4 vs. lane 1). Importantly, phosphorylation levels of IKKs and p65 were increased in A549 cells treated with LPS or CXCL13. They were markedly elevated in response to co-treatment of LPS and CXCL13 (Figures 3C–E). Consistently, NF-κB activity and production levels of IL-6 and IL-1β cytokines were significantly elevated in the group co-treated with LPS and CXCL13 (Figure 3F, NF-κB activity; Figure 3G, IL-6; Figure 3H, IL-1β). To determine whether the activation of NF-κB in CXCR5-KO lung cancer cells was affected, Ctrl A549, Ctrl H1299, CXCR5-KO A549 and CXCR5-KO H1299 cells were treated with LPS, CXCL13 or LPS plus CXCL13. Phosphorylation levels of IKKs and p65 were significantly attenuated in CXCR5-KO A549 and CXCR5-KO H1299 cells treated with LPS, CXCL13 or LPS plus CXCL13, as compared to those in Ctrl A549 and Ctrl H1299 cells (Figure 3I, Ctrl A549 and CXCR5-KO A549; Figure S7, Ctrl H1299 and CXCR5-KO H1299). Consistent results were observed in the NF-κB reporter assay (Figure S8A, Ctrl A549 and CXCR5-KO A549; Figure S8B, Ctrl H1299 and CXCR5-KO H1299). Moreover, the phosphorylation of AKT, which is involved in cell proliferation and survival, was markedly attenuated in CXCR5-KO A549 and CXCR5-KO H1299 cells treated with LPS, CXCL13 or LPS plus CXCL13, as compared with those in Ctrl A549 and Ctrl H1299 cells (Pho-AKT; Figures 3I and S7). These results suggest that CXCR5 and TLR4 signals can synergistically induce the activation of NF-κB and AKT for proliferation and survival (Figure 3J), thereby regulating lung cancer growth. Given the above results, we examined whether CXCR5 and TLR4 signals regulated lung cancer progression. Ctrl A549, Ctrl H1299, CXCR5-KO A549 and CXCR5-KO H1299 cells were treated with CXCL13, LPS or CXCL13 plus LPS. Wound healing assay and transwell migration assay revealed that CXCR5-KO A549 and CXCR5-KO H1299 cells showed reduced migration ability in response to CXCL13, LPS or CXCL13 plus LPS compared with Ctrl A549 and Ctrl H1299 cells (Figures 4A-H, CXCR5-KO A549 and CXCR5-KO H1299 vs. Ctrl A549 and Ctrl H1299). Moreover, CXCR5-KO A549 and CXCR5-KO H1299 cells treated with CXCL13, LPS or CXCL13 plus LPS showed significantly attenuated proliferation ability (Figure 4I, Ctrl A549 and CXCR5-KO A549; Figure 4J, Ctrl H1299 and CXCR5-KO H1299). Consistently, Ctrl A549 and Ctrl H1299 cells treated with CXCL13, LPS or CXCL13 plus LPS showed significantly enhanced anchorage-dependent and -independent colony formation ability, whereas CXCR5-KO A549 and CXCR5-KO H1299 cells showed marked attenuation of colony formation ability (Figures 4K and L, Ctrl A549 and CXCR5-KO A549; Figures 4M and N, Ctrl H1299 and CXCR5-KO H1299; Figures S9A and B, Ctrl A549 and CXCR5-KO A549; Figures S9C and D, Ctrl H1299 and CXCR5-KO H1299). We finally assessed whether the deficiency of CXCR5 is affected on tumour spheroid formation. We performed the 3D tumour spheroid assay with Ctrl A549 or CXCR5-KO A549 cells treated with vehicle, CXCL13, LPS or CXCL13 plus LPS. The spheroid size was increased in Ctrl A549 cells treated with CXCL13, LPS or CXCL13 plus LPS, as compared with those treated with vehicle (Figures 4O and P, Ctrl A549 treated with CXCL13, LPS or CXCL13 plus LPS vs. vehicle). Importantly, the spheroid size was significantly decreased in CXCR5-KO A549 cells treated with vehicle, CXCL13, LPS or CXCL13 plus LPS, as compared with those of Ctrl A549 cells (Figures 4O and P, CXCR5-KO A549 vs. Ctrl A549). In summary, our results demonstrate that the expression of CXCR5 in lung tumour tissues of NSCLC patients is associated with cancer progression. Up-regulated CXCR5, CXCL13 and TLR4 in lung tumour tissues were significantly enriched with gene sets regulating cancer formation and development, chemokine and innate signalling pathways. Importantly, CXCR5-KO human lung cancer cells exhibited marked attenuations of cancer migration, proliferation and colony formation ability in response to CXCL13. In terms of functional aspects, the expression of CXCR5 was up-regulated by TLR4 signalling through the activation of NF-κB. Therefore, the lung cancer progressive ability was significantly elevated in response to CXCL13 and LPS, but markedly attenuated in CXCR5-KO human lung cancer cells. As depicted in Figure 4Q, we propose a possible scenario in which the CXCR5–CXCL13 signalling axis is functionally implicated in lung cancer progression through a synergetic effect of TLR4 signalling. TLR4 signalling induces the production of CXCL13 and increases the expression of CXCR5 via activation of NF-κB.8 Notably, it has been reported that bacterial infection is a potent cancer-inducing factor that triggers cancer progression.10 Therefore, lung cancer patients with up-regulation of TLR4 and CXCR5 might be expected to be more likely to experience NSCLC progression if they have bacterial or viral infections (Figure 4Q, down). Furthermore, it has been reported that TLR4 is strongly expressed in lung cancer tissues and associated with cancer progression, along with poor prognosis of patients with NSCLC.7 In addition, CXC chemokine ligand-13 promotes metastasis via a CXCR5-dependent signalling pathway in NSCLC,2 indicating a promising target for the prevention and inhibition of metastasis. Taken together, our clinically comparative results and functional investigations suggest that CXCL13/CXCR5 and TLR4 signals might be potential therapeutic targets capable of intervening NSCLCs in terms of clinical and application aspects. E. C. and K. Y. L. designed and supervised all experiments and contributed to the manuscript preparation. J. H. S., M. J. K., J. Y. K., Y. K. and S. K. J. performed the experiments and analysed the data. D. H. K., E. C. and K. Y. L. analysed TCGA and microarray data and contributed to the manuscript preparation. E. C. and K. Y. L. wrote the manuscript. All authors have read and approved the final manuscript. We would like to thank Hyehwa Forum members for their helpful discussion. The authors declare that they have no competing interests. This work was supported by grants (2023R1A2C1003762, 2021R1A2C1094478 and RS-2023-00217189) of the National Research Foundation (NRF) funded by The Ministry of Science and ICT (MSIT), Republic of Korea. All authors agree to publish this article. All experiments were performed according to the Declaration of Helsinki and the study was approved by the Institutional Review Board (IRB) of Samsung Medical Center (SMC) (IRB#: 2010-07-204). Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Free fatty acid receptors (FFARs) and toll-like receptors (TLRs) recognize microbial metabolites and conserved microbial products, respectively, and are functionally implicated in inflammation and cancer. However, whether the crosstalk between FFARs and TLRs affects lung cancer progression has never been addressed. We analyzed the association between FFARs and TLRs using The Cancer Genome Atlas (TCGA) lung cancer data and our cohort of non-small cell lung cancer (NSCLC) patient data (n = 42), and gene set enrichment analysis (GSEA) was performed. For the functional analysis, we generated FFAR2-knockout (FFAR2KO) A549 and FFAR2KO H1299 human lung cancer cells and performed biochemical mechanistic studies and cancer progression assays, including migration, invasion, and colony-formation assays, in response to TLR stimulation. The clinical TCGA data showed a significant down-regulation of FFAR2, but not FFAR1, FFAR3, and FFAR4, in lung cancer, and a negative correlation with TLR2 and TLR3. Notably, GSEA showed significant enrichment in gene sets related to the cancer module, the innate signaling pathway, and the cytokine-chemokine signaling pathway in FFAR2DownTLR2UpTLR3Up lung tumor tissues (LTTs) vs. FFAR2upTLR2DownTLR3Down LTTs. Functionally, treatment with propionate (an agonist of FFAR2) significantly inhibited human A549 or H1299 lung cancer migration, invasion, and colony formation induced by TLR2 or TLR3 through the attenuation of the cAMP-AMPK-TAK1 signaling axis for the activation of NF-κB. Moreover, FFAR2KO A549 and FFAR2KO H1299 human lung cancer cells showed marked increases in cell migration, invasion, and colony formation in response to TLR2 or TLR3 stimulation, accompanied by elevations in NF-κB activation, cAMP levels, and the production of C-C motif chemokine ligand (CCL)2, interleukin (IL)-6, and matrix metalloproteinase (MMP) 2 cytokines. Our results suggest that FFAR2 signaling antagonized TLR2- and TLR3-induced lung cancer progression via the suppression of the cAMP-AMPK-TAK1 signaling axis for the activation of NF-κB, and its agonist might be a potential therapeutic agent for the treatment of lung cancer.

Dear editor, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can lead to severe outcomes in patients with cancer [1]. It has been reported that patients with lung cancers disproportionately manifest severe COVID-19 with a high rate of hospitalization and death [2]. Notably, the SARS-CoV-2 Spike (S) protein can induce hyper-inflammation in both epithelial cells and macrophages through toll-like receptor (TLR)1/TLR2 or TLR2/6-dependent nuclear factor-kappaB (NF-κB) pathway [3]. However, molecular and cellular evidence on whether the SARS-CoV-2 virus affects the severity of lung cancer patients through TLR1/2 or TLR2/6 signaling remains unclear. To obtain insight into the association of SARS-CoV-2 susceptibility and severity in lung cancer, we utilized microarray data of 42 non-small cell lung cancer (NSCLC) patients and analyzed different magnitude differences (∆Mag) of angiotensin converting enzyme 2 (ACE2) and TLR2 expression (termed ∆ACE2 and ∆TLR2), which are associated with SARS-CoV-2 infection and hyper-immune response [1-3], between lung tumor tissues and matched lung normal tissues (Supplementary Table S1). Based on ∆ACE2 and ∆TLR2, we selected 11 ACE2upTLR2up lung tumor tissues and 11 ACE2downTLR2down lung tumor tissues (Supplementary Figure S1A) and performed gene set enrichment analysis (GSEA) (https://www.gsea-msigdb.org) to determine whether expression levels of ACE2 and TLR2 were statistically associated with gene sets related to cancer, SARS-CoV-2 infection, and TLR signaling. Ten gene sets of cancer modules were significantly enriched in ACE2upTLR2up lung tumor tissues versus ACE2downTLR2down lung tumor tissues (Supplementary Figure S1B). Additionally, gene sets related to SARS-CoV-2 infection (Supplementary Figure S2A) or the TLR signaling pathway (Supplementary Figure S2B) were significantly enriched in ACE2upTLR2up lung tumor tissues. To verify these results in detail, we further analyzed ∆Mag of transmembrane protease serine subtype 2 (TMPRSS2), which contributes to virulence and pathogenesis of SARS-CoV-2 virus along with ACE2 [1, 2], TLR1, and TLR6, which are functionally formed as a TLR1/2 or TLR2/6 heterodimer [3], between lung tumor tissues and matched lung normal tissues (Figure 1A, Supplementary Table S1). Based on ∆ACE2, ∆TMPRSS2, ∆TLR1, ∆TLR2, and ∆TLR6, we further selected 4 ACE2upTMPRSS2upTLR1upTLR2upTLR6up lung tumor tissues and 7 ACE2downTMPRSS2downTLR1downTLR2downTLR6down lung tumor tissues (Figure 1A) and performed GSEA. Nine gene sets of cancer modules regulating cancer progression and development and 2 gene sets of cancer modules involving anti-apoptosis or inflammatory response were enriched in ACE2upTMPRSS2upTLR1upTLR2upTLR6up lung tumor tissues (Figure 1B, Supplementary Figure S3). SARS-CoV-2 spike protein induces lung cancer migration and invasion in a TLR2-dependent manner. (A) 42 lung tumor tissues of NSCLC patients were listed according to the ∆Mag of ACE2, TMPRSS2, TLR1, TLR2, and TLR6 expression in lung tumor tissues (LTTs) versus matched lung normal tissues (mLNTs). Eleven lung tumor tissues, four ACE2upTMPRSS2upTLR1upTLR2upTLR6up lung tumor tissues and seven ACE2downTMPRSS2downTLR1downTLR2downTLR6down lung tumor tissues, were selected for GSEA. The color scale indicates ∆Mag value. (B) GSEA was performed in four ACE2upTMPRSS2upTLR1upTLR2upTLR6up lung tumor tissues versus seven ACE2downTMPRSS2downTLR1downTLR2downTLR6down lung tumor tissues indicated in (A). Gene sets for four cancer modules are presented. (C-F) Gene sets for ACE2 expressing cells with SARS-CoV-2 infection (C), TLR cascades (D), IL-1R pathway (E), and TNF pathway (F) are presented along with heat maps showing differential gene expression patterns between four ACE2upTMPRSS2upTLR1upTLR2upTLR6up lung tumor tissues and seven ACE2downTMPRSS2downTLR1downTLR2downTLR6down lung tumor tissues. NES, nominal P-value, and FDR q-values are indicated in the inner panel. (G) A549 lung cancer cells were treated with vehicle, SARS-CoV-2 S protein, Pam3CSK4, or FSL-1 (up). The residual gap between migrating cells from the opposing wound edge is expressed as a percentage of the initial scraped area (± SD, n = 3 different plates) (down). (H) A549 lung cancer cells were treated with vehicle, SARS-CoV-2 S protein, Pam3CSK4, or FSL-1 for 24 h (left). The number of migrating cells was counted. Results are presented as mean ± SD of three independent experiments (right). (I) A549 lung cancer cells were stimulated with SARS-CoV-2 S protein, Pam3CSK4, or FSL-1. Phosphorylation levels of P65, IKKs, and ERK were measured by Western blotting. (J) TLR2-knockout (TLR2-KO) A549 cells were generated using CRISPR-Cas9 gene-editing method. TLR2 expression was verified by western blotting with anti-TLR2 and anti-GAPDH (control). (K) Ctrl A549 and TLR2-KO A549 cells were treated with vehicle, 3-MA, SARS-CoV-2 S protein, Pam3CSK4, or FSL-1 for different time periods (up). The residual gap between migrating cells from the opposing wound edge is expressed as a percentage of the initial scraped area (± SD, n = 3 different plates) (down). (L) Ctrl A549 and TLR2-KO A549 cells were treated with vehicle, 3-MA, SARS-CoV-2 S protein, Pam3CSK4, or FSL-1 for 24 h (left). The number of migrating cells was counted. Results are presented as mean ± SD of three independent experiments (right). (M-O) Ctrl A549 and TLR2-KO A549 cells were treated with vehicle, SARS-CoV-2 S protein, Pam3CSK4, or FSL-1 for 12 h. Production levels of IL-6 (M), IL-1β (N), and TNF-α (O) in culture supernatant were measured by ELISA. Results are presented as mean ± SD of three independent experiments. (P) A schematic model of how the SARS-CoV-2 virus critically affects the susceptibility to SARS-CoV-2 infection and the severity of SARS-CoV-2 infection in lung cancer patients with up-regulation of ACE2, TMPRSS2, TLR1, TLR2, and TLR6. *, P < 0.05; **, P < 0.01; and ***, P < 0.001. Abbreviations: SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TLR, toll-like receptor; NSCLC, non-small cell lung cancer; ∆Mag; magnitude difference; ACE2, angiotensin-converting enzyme 2; TMPRSS2, transmembrane protease serine subtype 2; LTTs, lung tumor tissues; mLNTs, matched lung normal tissues; GSEA, gene set enrichment analysis; IL-1R, interleukin-1 receptor; TNF, tumor necrosis factor; Pam3CSK4, tripalmitoyl-S-glycero-Cys-(Lys) 4; FSL-1, fibroblast stimulating lipopeptide 1; TLR2-KO, TLR2-knockout; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ELISA, enzyme-linked immunosorbent assay; IKK, inhibitor of nuclear factor-κB kinase; ERK, extracellular signal-regulated kinase; CRISPR-Cas9, clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9; IL-6, interleukin 6. Remarkably, gene sets upregulated in SARS-CoV-2 infection and innate signaling pathways, including TLR cascades and the nucleotide-binding and oligomerization domain (NOD)-like receptor pathway, were significantly enriched in ACE2upTMPRSS2upTLR1upTLR2upTLR6up lung tumor tissues versus ACE2downTMPRSS2downTLR1downTLR2downTLR6down lung tumor tissues (Figure 1C-D, Supplementary Figure S4A), along with their downstream signaling targets and pathways, such as the NF-κB pathway, NF-κB1 targets, tumor necrosis factor (TNF) signaling via NF-κB, interferon-α/β signaling, the interleukin-1 receptor (IL-1R) pathway, and the TNF pathway (Figure 1E-F, Supplementary Figure S4B-E). SARS-CoV-2 virus can infect human cells and induce inflammatory cytokines and chemokines, including IL-6, IL-1β, TNF-α, C-X-C motif chemokine ligand 1 (CXCL1), CXCL2, and C-C motif chemokine ligand 2 (CCL2), via TLR2-dependent activation of the NF-κB pathway [3-6]. Therefore, we further assessed whether expression levels of ACE2, TMPRSS2, TLR1, TLR2, and TLR6 were associated with gene sets related to inflammatory cytokines and chemokines. A gene set related to cytokine receptor interaction was highly enriched in ACE2upTMPRSS2upTLR1upTLR2upTLR6up lung tumor tissues versus ACE2downTMPRSS2downTLR1downTLR2downTLR6down lung tumor tissues (Supplementary Figure S5A). Gene sets related to signal transduction through IL-1R, tumor necrosis factor receptor 2 (TNFR2) pathway, TNF targets, and cytokine pathways were enriched in ACE2upTMPRSS2upTLR1upTLR2upTLR6up lung tumor tissues versus ACE2downTMPRSS2downTLR1downTLR2downTLR6down lung tumor tissues (Supplementary Figure S5B-G). Additionally, gene sets of the IL-8-C-X-C motif chemokine receptor 1 (CXCR1), IL-8-CXCR2, and cytokine- and chemokine-mediated signals were enriched in ACE2upTMPRSS2upTLR1upTLR2upTLR6up lung tumor tissues (Supplementary Figure S5H-M). These results suggest that upregulation of ACE2, TMPRSS2, TLR1, TLR2, and TLR6 in lung cancer tissues are associated with gene sets related to cancer progression, SARS-CoV-2 infection, and inflammatory responses. Given that the SARS-CoV-2 S protein can induce inflammation via TLR1/2- or TLR2/6-dependent activation of the NF-κB pathway [3], we investigated whether the SARS-CoV-2 S protein could induce lung cancer migration, invasion, colony formation, and cell proliferation via TLRs. Upon treatment with the SARS-CoV-2 S protein, Pam3CSK4 (an agonist of TLR1/2), or FSL-1 (an agonist of TLR2/6), migration and invasion abilities of A549 and H1299 lung cancer cells were significantly enhanced compared to those upon treatment with vehicle control (Figure 1G-H, Supplementary Figure S6). Moreover, colony-forming and cell proliferation assay revealed significant increases in response to the SARS-CoV-2 S protein, Pam3CSK4, or FSL-1 (Supplementary Figure S7). Notably, stimulation of the SARS-CoV-2 S protein, Pam3CSK4, or FSL-1 induced increases in phosphorylation of P65, inhibitor of nuclear factor-κB kinases (IKKs), and extracellular signal-regulated kinase (ERK) compared to treatment with vehicle in A549 cells (Figure 1I, Supplementary Figure S8A-C), accompanied with increases in activities of cytokines such as IL-6, IL-1β, TNF-α, and NF-κB (Supplementary Figure S8D-G). Consistent with data of A549 cells, H1299 cells treated with the SARS-CoV-2 S protein, Pam3CSK4, or FSL-1 showed elevated levels of phosphorylated P65, IKKs, and ERK (Supplementary Figure S9A-D) and production of IL-6, IL-1β, and TNF-α (Supplementary Figure S9E-G). These results suggest that the SARS-CoV-2 S protein can induce lung cancer progression by activating the NF-κB pathway via TLR1/2 and TLR2/6. To directly verify the functional role of the SARS-CoV-2 S protein in lung cancer progression through TLR1/2 and TLR2/6 stimulation, we generated TLR2-knockout (KO) A549 and H1299 cells using CRISPR-Cas9 gene-editing method [6-9] (Figure 1J, Supplementary Figure S10) and performed cancer progression assay as previously described [7-9]. Consistently, treatment with the SARS-CoV-2 S protein, Pam3CSK4, or FSL-1 enhanced cancer migration and invasion of control (Ctrl) A549 cells as compared to treatment with vehicle control, whereas TLR2-KO A549 cells showed markedly attenuated cancer migration and invasion (Figure 1K-L). On the other hand, 3-methyladenine (3-MA), an inhibitor of phosphoinositide-3-kinase (PI3K), inhibited cell migration and invasion of Ctrl A549 and TLR2-KO A549 cells (Figure 1K-L). Furthermore, TLR2-KO A549 cells treated with the SARS-CoV-2 S protein, Pam3CSK4, or FSL-1 showed significantly reduced production of IL-6, IL-1β, and TNF-α as compared to Ctrl A549 cells (Figure 1M-O). We observed similar results of cell migration and invasion of TLR2-KO H1299 cells (Supplementary Figure S11). As depicted in Figure 1P, these results suggest that the SARS-CoV-2 S protein can directly induce lung cancer progression, including migration, invasion, colony formation, and proliferation, in a TLR2-dependent manner. In conclusion, we provide evidence about how SARS-CoV-2 critically affects viral susceptibility and severity in patients with lung cancer. Our data demonstrate that lung cancer patients with up-regulated ACE2, TMPRSS2, TLR1, TLR2, and TLR6 are more likely to be susceptible to SARS-CoV-2 infection than those with down-regulated ACE2, TMPRSS2, TLR1, TLR2, and TLR6, subsequently leading to a more severe SARS-CoV-2 infection followed by promoting cancer progression through TLR2-dependent activation of NF-κB. However, it is still controversial of the role of TLR2 in lung tumor progression because TLR2 orchestrates a tumor suppressor response in early-stage lung cancer through the induction of cell-autonomous and non-cell-autonomous tumor suppressor responses. Although the precise molecular and cellular mechanisms by which TLR2 is functionally implicated in different stages of lung cancer is absolutely required, the current study gives insight into cellular and molecular mechanisms by which SARS-CoV-2 infection influences lung cancer progression in a TLR2-dependent manner. It might contribute to our understanding of the susceptibility to and the severity of SARS-CoV-2 infection in patients with lung cancer. EC and KYL conceptualized and designed the project; MJK, JYK, JHS, JS, YK, and SKJ performed experiments and analyzed data; EC, KYL, KHK, and DHK analyzed microarray data; EC and KYL wrote the manuscript; All authors read and approved the manuscript. We would like to thank Hyehwa Forum members for their helpful discussion. This work was supported by the National Research Foundation of Korea Grants funded by the Korean Government (2023R1A2C1003762, 2021R1A2C1094478, 2021M3A912080488, and RS-2023-00217189). Tumor and matched normal tissues from 42 patients with primary NSCLC were obtained in accordance with the ethical principles stated in the Declaration of Helsinki. This study was approved by the Institutional Review Board of Samsung Medical Center (IRB#: 2010-07-204). We obtained written informed consent from each patient prior to surgery for using their pathological specimens for research use. Not applicable The authors declare that they have no conflicts of interest. The data that support the findings of this study are available from the corresponding author upon reasonable request. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

β‐arrestin 2 (ARRB2) is functionally implicated in cancer progression via various signaling pathways. However, its role in lung cancer remains unclear. To obtain clinical insight on its function in lung cancer, microarray data from lung tumor tissues (LTTs) and matched lung normal tissues (mLNTs) of primary non-small cell lung cancer (NSCLC) patients (n = 37) were utilized. ARRB2 expression levels were markedly decreased in all 37 LTTs compared to those in matched LNTs of NSCLC patients. They were significantly co-related to enrichment gene sets associated with oncogenic and cancer genes. Importantly, Gene Set Enrichment Analysis (GSEA) between three LTTs with highly down-regulated ARRB2 and three LTTs with lowly down-regulated ARRB2 revealed significant enrichments related to toll-like receptor (TLR) signaling and autophagy genes in three LTTs with highly down-regulated ARRB2, suggesting that ARRB2 was negatively involved in TLR-mediated signals for autophagy induction in lung cancer. Biochemical studies for elucidating the molecular mechanism revealed that ARRB2 interacted with TNF receptor-associated factor 6 (TRAF6) and Beclin 1 (BECN1), thereby inhibiting the ubiquitination of TRAF6-TAB2 to activate NF-κB and TRAF6-BECN1 for autophagy stimulated by TLR3 and TLR4, suggesting that ARRB2 could inhibit the TRAF6-TAB2 signaling axis for NF-κB activation and TRAF6-BECN1 signaling axis for autophagy in response to TLR3 and TLR4. Notably, ARRB2-knockout (ARRB2KO) lung cancer cells exhibited marked enhancements of cancer migration, invasion, colony formation, and proliferation in response to TLR3 and TLR4 stimulation. Altogether, our current data suggest that ARRB2 can negatively regulate lung cancer progression by inhibiting TLR3- and TLR4-induced autophagy.

Dear Editor, Cereblon (CRBN) has been identified as a primary target of immunomodulatory drugs in multiple myeloma.1 Herein, for the first time, we demonstrate that CRBN expression is functionally involved in lung cancer progression through the regulation of autophagy by toll-like receptor (TLR)2, TLR4 and TLR7. TLR signalling is associated with the induction of autophagy and plays a pivotal role in the progression and pathogenesis of lung cancer.2 Our study suggests that CRBN can be a potent prognostic marker for lung cancer and provides important implications in clinical and translational lung cancer biology. To get insight into the association of CRBN with lung cancer, we utilized data from the TCGA (The Cancer Genome Atlas) (GEPIA, http://gepia.cancer-pku.cn/detail.php?gene = CRBN) and a cohort of NSCLC patients (n = 18). GEPIA database revealed that CRBN was downregulated in lung adenocarcinoma and lung squamous cell carcinoma (Figures S1 and 1A, LUSC, *p < .05). Moreover, CRBN was downregulated in lung tumour tissues (LTTs) of 18 NSCLC patients compared to matched lung normal tissues (LNTs) (Figure 1B and, Table S1). To see whether CRBN expression is associated with a defined set of genes related to cancer in LTTs of NSCLC patients, we selected the top seven LTTs (<−1.5-fold decrease of CRBN) (Figure 1B, red bars) and performed gene set enrichment analysis (GSEA). The 15 enrichment gene set plot related to oncogenic signature and cancer was enriched in the top 7 LTTs as compared to matched LNTs of NSCLC patients (Figure S2). To decipher the gene association between CRBN expression and lung cancer progression in the 7 LTTs, we aligned 500 downregulated or upregulated genes based on the data of LTT36 with the most downregulation (Figure S3A and Table S2, downregulated genes; Figure S4A and Table S3, upregulated genes). We further sorted downregulated or upregulated genes in all seven LTTs (Figures S3B–D and S4B,C). Then, we accessed PubMed to confirm whether these genes have been reported in lung cancer proliferation, migration or invasion and progression. Among downregulated genes (Figure S3B–D), 17, 11 and 21 genes have been involved in anti-lung cancer proliferation (Figure 1C, Table S4), anti-lung cancer migration or invasion (Figure 1D, Table S5) and anti-lung cancer progression (Figure 1E, Table S6), respectively. Among upregulated genes (Figures 1F and S4B,C), 13, 10 and 4 genes have been involved in lung cancer proliferation (Figure 1G, Table S7), migration or invasion (Figure 1H, Table S8) and progression (Figure 1I, Table S9), respectively. We observed a similar pattern in the other 11 LTTs (Figures S5A–C and S5D–F). To examine the role of CRBN in lung cancer cells, we generated CRBN-knockout (CRBNKO) A549 (Figure 1J) and H1299 lung cancer cells (Figure S6) and performed in vitro and vivo cancer progression assays. CRBNKO A549 cells significantly enhanced cancer migration (Figure 1K), invasion (Figure 1L), single-cell migration (Figure 1 M), anchorage-dependent (Figure 1N) and anchorage-independent colony formation (Figure 1O). We also observed a similar pattern in CRBNKO H1299 cells (Figure S7A–D). Notably, as an in vivo model, NOD/SCID/IL2rγnull (NSG) mice engrafted with CRBNKO A549 cells markedly increased the tumour sizes and masses (Figure 1P,Q, CRBNKO A549 vs. Ctrl A549). H&E staining data revealed that the number of neoplastic epithelial cells was higher in tumour and metastasized LTTs derived from mice injected with CRBNKO A549 (Figure S8A,B, tumour; Figure S8C,D, metastasized lung tumour) compared to those of Ctrl A549, demonstrating that (i) CRBN downregulation is associated with lung cancer; (ii) CRBN-deficiency promotes in vitro and vivo lung cancer progression. Growing evidence suggests that intrinsic and extrinsic factors, including spontaneous mutations of genes and TLRs, play pivotal roles in lung cancer development and progression.2-7 Importantly, TLR2, TLR4 and TLR7 are expressed in lung cancer and associated with lung cancer progression.5-7 Additionally, TLR4- and TLR3- induced autophagy facilitates migration and invasion of lung cancer cells.2 We previously reported that CRBN negatively regulates TLR4 signalling through the attenuation of ubiquitination of TRAF6, and autophagy activation by inhibiting the ubiquitination of BECN1.8, 9 Therefore, we sought to determine whether CRBN is involved in lung cancer progression by TLR2, TLR4 and TLR7 signals associated with autophagy. We first examined if TLR2, TLR4 and TLR7 signals are associated with genes related to lung cancer progression and autophagy. We treated A549 and H1299 cells with vehicle as a control, TLR2 (heat-killed Listeria monocytogenes, HKLM), TLR4 (lipopolysaccharide, LPS) or TLR7 (imiquimod, IQM) agonists and performed RNA-sequencing analysis. The GSEA of transcriptional profiles revealed that 15 enrichment gene set plots related to cancer gene neighbourhoods and cancer modules, ontology and oncogenic signature were enriched in A549 and H1299 treated with TLR4 agonist compared to those with the vehicle (Figure S9A–C, A549; Figure S9D–F, H1299). To examine the association of genes related to lung cancer progression and autophagy in response to TLR stimulation, we arranged 500 upregulated genes (Figure S10A and Table S10 in A549; Figure S10B, Table S11 in H1299) or 500 downregulated genes (Figure S10C and Table S12 in A549; Figure S10D and Table S13 in H1299) based on the data of TLR4 stimulation. Then, we further sorted upregulated (Figure S11A and Table S14 in A549; Figure S11B and Table S15 in H1299) or downregulated (Figure S11C and Table S16 in A549; Figure S11D and Table S17 in H1299) genes in all three TLR stimulations. Among upregulated genes, 12 genes (Figure 2A and Table S18) in A549 cells and 21 genes (Figure 2B and Table S19) in H1299 cells have been involved in lung cancer progression. In the list of downregulated genes, 10 genes (Figure 2C and Table S20) in A549 cells and 11 genes (Figure 2D and Table S21) in H1299 cells have been involved in anti-lung cancer progression. Notably, 13 genes upregulated in A549 or H1299 cells have been functionally associated with autophagy (Figure 2E, Table S22). Moreover, enrichment gene sets regulating autophagy, such as BECN1 (Figure S12A), MTOR (Figure S12B) and AKT-MTOR (Figure S12C) were enriched in A549 treated with TLR4 agonist compared to those with the vehicle. These results suggest that TLR2, TLR4 and TLR7 stimulation increase gene signatures related to lung cancer progression and autophagy (Figure 2F). We next examined whether CRBN is functionally involved in lung cancer progression through the regulation of autophagy by TLR stimulation. Under the TLR2 (HKLM), TLR4 (LPS) or TLR7 (IQM) stimulation, cell migration and invasion were significantly enhanced in CRBNKO A549 cells (Figure 2G,H, HKLM; Figure 2I,J, LPS; Figure 2K,L, IQM: CRBNKO A549 vs. Ctrl A549). Marked inhibition was observed in the presence of autophagy inhibitors, 3-MA or CQ (Figure 2G–L, HKLM, LPS or IQM agonist + 3-MA or CQ vs. TLR agonist alone). Importantly, LC3-II levels and LC3 puncta that represent induction of autophagy were significantly enhanced in CRBNKO A549 treated with TLR agonists (Figure 3A–C, LC3-II levels; Figure 3D,E, LC3 puncta). TLR4 signalling induces autophagy through the BECN1 ubiquitination by TRAF6.2, 8-10 Then, we examined whether CRBN-deficiency directly affects the BECN1 ubiquitination by TLR2, TLR4 and TLR7 stimulation. BECN1 ubiquitination was markedly elevated in CRBNKO A549 cells treated with HKLM, LPS or IQM as compared to those of Ctrl A549 cells (Figure 3F, lane 6 vs. lane 2 in HKLM; lane 7 vs. lane 3 in LPS; lane 8 vs. lane 4 in IQM). IL-6, CCL2, CCL20 and MMP2 productions are necessary for enhanced migration and invasion of lung cancer cells upon TLR activation.2 We found that CRBNKO A549 cells markedly increased IL-6, CCL2, CCL20 and MMP2 in response to three TLR agonists compared to those of Ctrl A549 (Figure 3G, IL-6; Figure 3H, CCL2; Figure 3I, CCL20; Figure 3J, MMP2: CRBNKO A549 vs. Ctrl A549), whereas marked inhibitions were observed in the co-treatment of autophagy inhibitors (Figure 3G–J, 3-MA). Lastly, we found that CRBNKO A549 cells treated with TLR agonists significantly increased single-cell mobility and the number of colonies compared to those of Ctrl A549 cells (Figure 4A,B, single-cell mobility; Figure 4C,D, number of colonies). In summary, CRBN is downregulated in lung cancer cells and associated with lung cancer progression (Figure 4E, upper right). Our study demonstrates the association between TLR stimulation and gene signatures related to lung cancer progression and autophagy. CRBN inhibits the BECN1 ubiquitination to induce autophagy and attenuates the production of IL-6, CCL2, CCL20 and MMP2 cytokines in response to TLR stimulations in healthy lung cells expressing CRBN (CRBNhigh, Figure 4E, down left). In lung cancer cells with downregulated CRBN (CRBNlow, Figure 4E, down right), engagements of TLRs enhance autophagy induction through the increases of BECN1 ubiquitination and the production of IL-6, CCL2, CCL20 and MMP2 cytokines, eventually facilitating lung cancer progression. Taken together, our clinically comparative results and functional investigations of CRBN in lung cancer progression will potentially contribute to translational approaches for lung cancer intervention. We would like to thank Hyehwa Forum members for their helpful discussion. This work was supported by the National Research Foundation of Korea (NRF) Grants funded by the Korean Government (NRF-2021R1F1A1049324 and NRF-2021R1A2C1094478), Ministry of Science ICT and Future Planning (MSIP) funded by the Korean Government (NRF-2016R1A5A2945889). The authors declare that they have no conflict of interest. All authors agree to publish this article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Lung cancer progression is regulated by various extrinsic factors derived from tumour microenvironment as well as intrinsic factors.1 Recent studies have shown that toll-like receptors (TLRs) are expressed in lung cancers, suggesting that TLRs may be implicated in lung cancer progression.2, 3 Although several studies have shown that stratifin (SFN, 14-3-3 sigma) facilitated lung cancer development and progression,4-6 the molecular and cellular mechanisms by which SFN is functionally involved in lung cancer progression, and the role of SFN in lung cancer progression in response to extrinsic stimulation, such as TLR agonist, are largely unknown. Here, we show that SFN expression is remarkably up-regulated in lung cancer tissues through The Cancer Genome Atlas (TCGA) data and primary non-small cell lung cancers (n = 31 of our cohort patients) analysis, and SFN positively regulates lung cancer progression through the autophagy induction by facilitating TRAF6- Vps34-BECN1 complex in response to an extrinsic TLR4 agonist. To get insight into the role of SFN in cancers, we first investigated whether the expression of SFN is associated with 33 different cancer types by analyzing TCGA datasets (http://gepia.cancer-pku.cn/detail.php?gene=SFN, Figure 1A). The gene expression of SFN was significantly up-regulated in 17 different tumour samples (Figure 1A, tumours are marked in red; Figure S1), whereas significantly down-regulation was observed in four different tumour samples (Figure 1A, tumours are marked in green; Figure S2). In lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) tumour samples, SFN expression was significantly increased compared to those of paired normal tissues (Figure 1B,C). Interestingly, the percentage of survival was significantly lower in LUAD patients with high expression of SFN (Figure 1D, red vs. blue; p = .00065), whereas there was no significant difference was observed in LUSC patients (Figure 1E, red vs. blue; p = .74). Notably, microarray data analysis showed that the expression of SFN was increased in primary 23 LUAD patients and eight LUSC patients (Figure 1F), indicating that SFN may be functionally associated with LUAD. Given the above results, we examined whether the expression of SFN is associated with gene expressions related to cancer progression in primary LUAD patient tissues. We selected seven primary LUAD patients with high expression of SFN (Figure 1F, red bars), and the pathological classification was confirmed by H&E staining in matched lung normal tissue and lung tumour tissue, patient #26,7 #52, #13, #17, #51, #12 and #29 (Figure 2A). To sort out genes related to cancer progression, 500 up-regulated genes or 500 down-regulated genes were arranged based on the data of patient #26 with the most up-regulation of SFN among the seven primary LUAD patients (Figure 2B, 500 up-regulated genes; Table S1: Figure 2C, 500 down-regulated genes; Table S2). Among the 500 down-regulated genes, commonly up-regulated genes (Figure 2D and Table S3) or down-regulated genes (Figure 2E and Table S4) were further sorted out. Interestingly, 23 genes related to cancer proliferation (Figure 2F and Table S5), 17 genes related to cancer migration or invasion (Figure 2G and Table S6) and 12 genes related to cancer metastasis (Figure 2H and Table S7) were up-regulated in the seven primary LUAD patient tumour tissues. Notably, 12, 10 and 8 genes were functionally associated with lung cancer proliferation, migration or invasion and metastasis, respectively (Figure 2F–H, red boxes; Table S5-S7, red letters). Although the effect of SFN on cell cycle progression is controversial, a previous report has demonstrated that SFN in lung adenocarcinoma cells might have tissue-specific functions and regulate cell cycle progression in a positive manner.4 Importantly, we found that 12 genes related to lung cancer proliferation have been reported to be involved in promoting cell cycle progression (Figure S3), indicating that SFN expression is associated with genes related to cell cycle progression in cancers. Moreover, 34 cancer suppressors genes were down-regulated in tumour tissues of the seven primary LUAD patients (Figure 2I, Table S8), and 18 genes were functionally associated with lung cancer (Figure 2I, green box; Table S8, green letters). To verify the function of SFN in lung cancer progression, we generated SFN-knockout (SFNKO) A549 human adenocarcinoma cells using the CRISPR cas9 gene editing method (Figure 2J, lane 2). The SFNKO A549 cells revealed marked inhibitions of cancer invasion and migration and colony formation and single-cell mobility and proliferation as compared to those of control (Ctrl) A549 cells (Figure 2K, invasion; Figure 2L, migration; Figure 2M, anchorage-dependent colony formation; Figure 2N, anchorage-independent colony formation; Figure S4A,B, single-cell mobility; Figure S5, cell proliferation: SFNKO A549 vs. Ctrl A549). Based on the TCGA and primary LUAD patient data analysis and the cancer progression assay of SFNKO lung cancer cells, we suggested that SFN might be functionally implicated in lung cancer progression. Further, we explored the molecular mechanism through which SFN is implicated in lung cancer progression. It has been reported that SFN accelerates lung cancer progression by regulating cell proliferation,4 and TLRs expressed in lung cancer promote the growth of cancer cells by inducing cell proliferation.3 Upon extrinsic TLR3/4 stimulations in lung cancer, additionally, autophagy was induced through the TRAF6-BECN1 signaling axis, leading to enhanced cancer migration and invasion.8 In this study, we hypothesized that SFN regulates lung cancer progression through the autophagy induced by extrinsic TLR stimulation. SFN interacted with TRAF6 (Figure 3A, lane 4) or BECN1 (Figure 3B, lane 4). SFN interacted with TRAF6 wild type (WT), and TRAF6 110-522 and TRAF6 260-522 and TRAF6 349-522 truncated mutants (Figure 3C). To verify the interaction, we performed an additional IP experiment with Flag-TRAF6 1-349 and Flag-TRAF6 349-522 truncated mutants. SFN interacted with Flag-TRAF6 349-522 but not with TRAF6 1-349 (Figure S6), indicating that SFN interacts with the tumor necrosis factor receptor-associated factor-C terminus (TRAF-C) domain of TRAF6 (Figure 3C, down). Additionally, SFN interacted with BECN1 WT, but not with BECN1 1-269 and BECN1 1-127 truncated mutants, indicating that SFN interacts with the C-terminal domain of BECN1 (Figure 3D). These results suggest that SFN can nucleate the association of TRAF6-BECN1 (Figure 3E). Importantly, the ubiquitination of BECN1 was markedly enhanced in the presence of SFN as compared to the absence of SFN (Figure 3F, lane 4 and 5 vs. lane 3), indicating that SFN nucleates the molecular association of TRAF6-BECN1 and enhances the ubiquitination of BECN1 (Figure 3G). Since Vps34 interacted with the coiled-coil domain of BECN1 and regulated autophagy,9 we further assessed whether SFN affects the association of Vps34-BECN1 complex. Our results showed that Vps34 interacted with SFN (Figure 3H, lane 4) or BECN1 (Figure 3I, lane 4). Furthermore, the interaction between Vps34 and BECN1 was markedly enhanced in the presence of SFN as compared to the absence of SFN (Figure 3J, lane 4 or 5 vs. lane 3). Consistently, the endogenous interaction between BECN1 and Vps34 was significantly decreased in SFNKO A549 cells in the presence or absence of lipopolysaccharide (LPS) as compared to those of Ctrl A549 cells (Figure S7, lane 3 and 4 in SFNKO A549 vs. lane 1 and 2 in Ctrl A549). Interestingly, the ubiquitination of BECN1 was significantly enhanced in the presence of SFN and Vps34 compared to their absence (Figure 3K, lane 4 vs. lane 3). These results suggest that SFN facilitates the molecular associations of the TRAF6-BECN1-Vps34 complex through the interaction with BECN1 (Figure 3D) or Vps34 (Figure 3H), leading to the enhancement of the BECN1 ubiquitination (Figure 3L). Autophagy regulated by the ubiquitination of BECN1 enhanced lung cancer migration and invasion in response to TLR3/4 stimulation.8 Upon TLR4 stimulation with LPS, the level of light chain 3-II (LC3-II) was significantly attenuated in SFNKO A549 cells as compared to Ctrl A549 (Figure 4A, lane 6 vs. lane 2). Moreover, cancer migration and invasion were markedly inhibited in SFNKO A549 cells treated with LPS, as compared to those of Ctrl A549 cells (Figure 4B,C, migration; Figure 4D,E, invasion: SFNKO A549 treated with LPS vs. Ctrl A549 treated with LPS). Consistently, single-cell mobility was significantly attenuated in SFNKO A549 cells treated with LPS (Figure S8A,B, SFNKO A549 vs. Ctrl A549). Matrix metalloproteinase-2 (MMP2) and interleukin-6 (IL-6), which are known to regulate cancer migration and invasion,8 were significantly down-regulated in SFNKO A549 cells treated with LPS as compared to Ctrl A549 cells treated with LPS (Figure 4F, MMP2; Figure 4G, IL-6). Furthermore, anchorage-dependent or -independent colony formation revealed a significant decrease in SFNKO A549 cells-treated LPS as compared to Ctrl A549 treated LPS (Figure 4H,I, anchorage-dependent; Figure 4J,K, anchorage-independent: SFNKO A549 cells treated LPS vs. Ctrl A549 cells treated LPS). Consistently, cell proliferation assay showed a significant decrease in SFNKO A549 cells in the presence of LPS (Figure S9, SFNKO A549 vs. Ctrl A549). In contrast, the co-treatment of autophagy inhibitors, 3-methyladenine (3-MA) and chloroquine (CQ), markedly attenuated cancer migration and invasion, and colony formation induced by LPS stimulation (Figure 4B–E,H–K, LPS vs. LPS + 3-MA or LPS + CQ). To verify these results, we performed rescue experiments with SFNKO A549 and SFNKO A549 transiently expressed with SFN (Figure S10). In SFNKO A549 cells transfected with hemagglutinin (HA)-SFN (Figure S10A, lane 2), cancer migration and invasion were significantly elevated as compared to those of SFNKO A549 cells transfected with mock vector (Figure S10B–C, HA-SFN-expressed SFNKO A549 vs. SFNKO A549). These results suggest that SFN positively regulates cancer migration and invasion, and colony formation through the autophagy induction by TLR4. In summary, we demonstrated that SFN expression in lung cancer is clinically associated with poor patient survival and lung cancer progression, accompanying the up-regulation of genes related to cancer progression and the down-regulation of genes related to cancer suppression. Through the biochemical and cellular studies, we propose possible molecular and cellular mechanisms in which SFN is functionally implicated in lung cancer progression: (1) SFN nucleates the TRAF6-BECN1-Vps34 complex and enhances the ubiquitination of BECN1 and subsequently regulates autophagy, (2) upon extrinsic TLR4 stimulation, SFN enhances cancer progression including cancer migration and invasion, proliferation and colony formation through autophagy induction. Together, our clinically comparative results and functional investigations of SFN expression in lung cancer will potentially contribute to translational approaches for the development of lung cancer therapeutic agents. The authors declare that they have no competing interests. National Research Foundation of Korea (NRF) Grants, Korean Government, Grant Numbers: NRF-2021R1F1A1049324 and NRF-2021R1A2C1094478; Korea Basic Science Institute (National Research Facilities and Equipment Center) Grant, Ministry of Education, Grant Number: 2020R1A6C101A191; Ministry of Science ICT and Future Planning (MSIP), Korean Government, Grant Number: NRF-2016R1A5A2945889 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

TNF receptor-associated factor 6 (TRAF6)-BECN1 signaling axis plays a pivotal role in autophagy induction through ubiquitination of BECN1, thereby inducing lung cancer migration and invasion in response to toll-like receptor 4 (TLR4) stimulation. Herein, we provide novel molecular and cellular mechanisms involved in the negative effect of ubiquitin-specific peptidase 15 (USP15) on lung cancer progression. Clinical data of the TCGA and primary non-small cell lung cancer (NSCLC) patients (n = 41) revealed that the expression of USP15 was significantly downregulated in lung cancer patients. Importantly, USP15-knockout (USP15KO) A549 and USP15KO H1299 lung cancer cells generated with CRISPR-Cas9 gene-editing technology showed increases in cancer migration and invasion with enhanced autophagy induction in response to TLR4 stimulation. In addition, biochemical studies revealed that USP15 interacted with BECN1, but not with TRAF6, and induced deubiquitination of BECN1, thereby attenuating autophagy induction. Notably, in primary NSCLC patients (n = 4) with low expression of USP15, 10 genes (CCNE1, MMP9, SFN, UBE2C, CCR2, FAM83A, ETV4, MYO7A, MMP11, and GSDMB) known to promote lung cancer progression were significantly upregulated, whereas 10 tumor suppressor genes (FMO2, ZBTB16, FCN3, TCF21, SFTPA1B, HPGD, SOSTDC1, TMEM100, GDF10, and WIF1) were downregulated, providing clinical relevance of the functional role of USP15 in lung cancer progression. Taken together, our data demonstrate that USP15 can negatively regulate the TRAF6-BECN1 signaling axis for autophagy induction. Thus, USP15 is implicated in lung cancer progression.

Herein, we aimed to elucidate the molecular and cellular mechanism in which ubiquitin-specific protease 8 (USP8) is implicated in liver cancer progression via TRAF6-mediated signal. USP8 induces the deubiquitination of TRAF6, TAB2, TAK1, p62, and BECN1, which are pivotal roles for NF-κB activation and autophagy induction. Notably, the LIHC patient with low USP8 mRNA expression showed markedly shorter survival time, whereas there was no significant difference in the other 18-human cancers. Importantly, the TCGA data analysis on LIHC and transcriptome analysis on the USP8 knockout (USP8KO) SK-HEP-1 cells revealed a significant correlation between USP8 and TRAF6, TAB2, TAK1, p62, and BECN1, and enhanced NF-κB-dependent and autophagy-related cancer progression/metastasis-related genes in response to LPS stimulation. Furthermore, USP8KO SK-HEP-1 cells showed an increase in cancer migration and invasion by TLR4 stimulation, and a marked increase of tumorigenicity and metastasis in xenografted NSG mice. The results demonstrate that USP8 is negatively implicated in the LIHC progression through the regulation of TRAF6-mediated signal for the activation of NF-κB activation and autophagy induction. Our findings provide useful insight into the LIHC pathogenesis of cancer progression.

Hepatitis B virus X (HBx) protein has been reported as a key protein regulating the pathogenesis of HBV-induced hepatocellular carcinoma (HCC). Recent evidence has shown that HBx is implicated in the activation of autophagy in hepatic cells. Nevertheless, the precise molecular and cellular mechanism by which HBx induces autophagy is still controversial. Herein, we investigated the molecular and cellular mechanism by which HBx is involved in the TRAF6-BECN1-Bcl-2 signaling for the regulation of autophagy in response to TLR4 stimulation, therefore influencing the HCC progression. HBx interacts with BECN1 (Beclin 1) and inhibits the association of the BECN1-Bcl-2 complex, which is known to prevent the assembly of the pre-autophagosomal structure. Furthermore, HBx enhances the interaction between VPS34 and TRAF6-BECN1 complex, increases the ubiquitination of BECN1, and subsequently enhances autophagy induction in response to LPS stimulation. To verify the functional role of HBx in liver cancer progression, we utilized different HCC cell lines, HepG2, SK-Hep-1, and SNU-761. HBx-expressing HepG2 cells exhibited enhanced cell migration, invasion, and cell mobility in response to LPS stimulation compared to those of control HepG2 cells. These results were consistently observed in HBx-expressed SK-Hep-1 and HBx-expressed SNU-761 cells. Taken together, our findings suggest that HBx positively regulates the induction of autophagy through the inhibition of the BECN1-Bcl-2 complex and enhancement of the TRAF6-BECN1-VPS34 complex, leading to enhance liver cancer migration and invasion.

TRAF6-BECN1 signaling axis is critical for autophagy induction and functionally implicated in cancer progression. Here, we report that AMP-activated protein kinase alpha 1 (AMPKα1, PRKAA1) is positively involved in autophagy induction and cancer progression by regulating TRAF6-BECN1 signaling axis. Mechanistically, AMPKα1 interacted with TRAF6 and BECN1. It also enhanced ubiquitination of BECN1 and autophagy induction. AMPKα1-knockout (AMPKα1KO) HEK293T or AMPKα1-knockdown (AMPKα1KD) THP-1 cells showed impaired autophagy induced by serum starvation or TLR4 (Toll-like receptor 4) stimulation. Additionally, AMPKα1KD THP-1 cells showed decreases of autophagy-related and autophagosome-related genes induced by TLR4. AMPKα1KO A549 cells exhibited attenuation of cancer migration and invasion induced by TLR4. Moreover, primary non-small cell lung cancers (NSCLCs, n = 6) with low AMPKαl levels showed markedly decreased expression of genes related to autophagy, cell migration and adhesion/metastasis, inflammation, and TLRs whereas these genes were significantly upregulated in NSCLCs (n = 5) with high AMPKαl levels. Consistently, attenuation of cancer migration and invasion could be observed in AMPKα1KO MDA-MB-231 and AMPKα1KO MCF-7 human breast cancer cells. These results suggest that AMPKα1 plays a pivotal role in cancer progression by regulating the TRAF6-BECN1 signaling axis for autophagy induction.

Toll-like receptors (TLRs) induce the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and autophagy through the TNF (Tumor necrosis factor) receptor-associated factor 6 (TRAF6)-evolutionarily conserved signaling intermediate in Toll pathways (ECSIT) and TRAF6-BECN1 signaling axes, respectively. Having shown that p62 negatively regulates Toll-like receptor 4 (TLR4)-mediated signaling via TRAF6-ECSIT signaling axis, we herein investigated whether p62 is functionally implicated in the TRAF6-BECN1 signaling axis, thereby regulating cancer cell migration and invasion. p62 interacted with TRAF6 and BECN1, to interrupt the functional associations required for TRAF6-BECN1 complex formation, leading to inhibitions of BECN1 ubiquitination and autophagy activation. Importantly, p62-deficient cancer cells, such as p62-knockdown (p62KD) SK-HEP-1, p62KD MDA-MB-231, and p62-knockout (p62KO) A549 cells, showed increased activation of autophagy induced by TLR4 stimulation, suggesting that p62 negatively regulates autophagy activation. Moreover, these p62-deficient cancer cells exhibited marked increases in cell migration and invasion in response to TLR4 stimulation. Collectively, these results suggest that p62 is negatively implicated in the TRAF6-BECN1 signaling axis, thereby inhibiting cancer cell migration and invasion regulated by autophagy activation in response to TLR4 stimulation.

Cereblon (CRBN) as a multifunctional protein has been extensively studied. Here, we show that CRBN is a negative regulator of bactericidal activity and autophagy activation. Mitochondrial localization of CRBN was significantly increased in response to Toll-like receptor 4 (TLR4) stimulation. CRBN interrupted the association of evolutionarily conserved signaling intermediate in Toll pathways (ECSIT)-TNF-receptor associated factor 6 (TRAF6) complex, thereby inhibiting the ubiquitination of ECSIT, which plays a pivotal role for the production of mitochondrial reactive oxygen species (mROS). Subsequently, mROS levels were markedly elevated in CRBN-knockdown (CRBNKD) THP-1 cells, and that led to resistance against S. typhimurium infection, indicating CRBN is a negative regulator of bactericidal activity through the regulation of mROS. Additionally, CRBN inhibited TRAF6-induced ubiquitination of BECN1 (Beclin 1), and that induced autophagy activation in CRBNKD THP-1, CRBN-knockout (CRBNKO) H1299, and CRBNKO MCF7 cancer cells in response to TLR4 stimulation. Notably, we found that the ability of cancer migration and invasion was significantly enhanced in CRBNKO H1299 and CRBNKO MCF7 cancer cells, as compared with those of control cancer cells. Collectively, these results suggest that CRBN is a negative regulator of bactericidal activity and autophagy activation through inhibiting the TRAF6-induced ubiquitination of ECSIT and BECN1, respectively.