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.

Sequestosome 1 (SQSTM1, p62), a ubiquitin binding protein, plays a role in cell signaling, oxidative stress, and autophagy. However, its functional role in inflammatory signaling is controversial. Recent studies have shown that p62 is negatively implicated in inflammatory responses. But, the precise molecular mechanisms by which p62 regulates inflammatory responses remain unclear. In this study, we report on a new regulatory role for p62 in TLR4-mediated signaling. p62 overexpression led to the suppression of NF-κB activation and the production of pro-inflammatory cytokines, TNF-α, IL-6, and IL-1β in response to TLR4 stimulation. In contrast, p62 -/- mouse embryonic fibroblast (MEF) cells exhibited marked enhancement of NF-κB activation and production of pro-inflammatory cytokines by TLR4 stimulation, compared to p62 +/+ MEF cells. Additionally, the TLR4-induced activation of signal transduction was significantly augmented in p62 -/- MEF cells, indicating that p62 was negatively implicated in TLR4-mediated signaling. Biochemical studies revealed that p62 interacted with the internal domain of evolutionarily conserved signaling intermediate in Toll pathways (ECSIT), which is critical for associating with the TNF receptor associated factor 6 (TRAF6)-ECSIT complex to activate NF-κB in TLR4 signaling. Interestingly, p62-ECSIT interaction inhibited the interaction between TRAF6 and ECSIT and attenuated the ubiquitination of ECSIT. Furthermore, upon LPS challenge, the mortality of p62 -/- (p62-knockout) mice was markedly enhanced compared to p62 +/+ (p62 wild-type) mice. Taken together, our data demonstrate that p62 negatively regulated TLR4 signaling via functional regulation of the TRAF6-ECSIT complex.

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.

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.