cells

Article p62 is Negatively Implicated in the TRAF6-BECN1 Signaling Axis for Activation and Cancer Progression by Toll-Like Receptor 4 (TLR4)

1, 1, 1, 1 2,3 Mi-Jeong Kim †, Yoon Min †, Ji Seon Im †, Juhee Son , Joo Sang Lee and Ki-Young Lee 1,3,4,*

1 Department of Immunology, Sungkyunkwan University School of Medicine, 2066 Seobu-ro, Jangan-gu, Suwon, Gyeonggi-do 16419, Korea; [email protected] (M.-J.K.); [email protected] (Y.M.); [email protected] (J.S.I.); [email protected] (J.S.) 2 Department of Precision Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, 2066 Seobu-ro, Jangan-gu, Suwon, Gyeonggi-do 16419, Korea; [email protected] 3 Samsung Medical Center, Seoul 06351, Korea 4 Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences & Technology, Samsung Medical Center, Sungkyunkwan University, 81 Irwon-ro, Gangnam-gu, Seoul 06351, Korea * Correspondence: [email protected]; Tel.: +82-31-299-6225 Authors contributed equally to this work. †  Received: 26 March 2020; Accepted: 2 May 2020; Published: 6 May 2020 

Abstract: 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 (Tumornecrosis 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.

Keywords: toll-like receptor 4; p62; TRAF6; BECN1; Autophagy

1. Introduction p62 (SQSTM1 and sequestosome-1) is a ubiquitin-binding and a versatile adaptor protein with multiple functions for regulating cellular events [1,2]. These include autophagic flux through the interaction with autophagic substrates, apoptosis, cellular redox regulation through the Kelch-like ECH-associated protein 1-nuclear factor (erythroid-derived 2)-like 2 (KEAP1-NRF2) pathway, adipogenesis by the interaction with extracellular-signal-regulated kinase 1 (ERK1), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling through the interaction with

Cells 2020, 9, 1142; doi:10.3390/cells9051142 www.mdpi.com/journal/cells Cells 2020, 9, 1142 2 of 14 protein kinase C ζ (PKCζ)[1–8]. Although the regulatory role of p62 in inflammatory responses is controversial [9,10], it is thought to be involved in the induction of inflammatory cytokine production via TNF Tumor necrosis factor) receptor-associated factor 6 (TRAF6) polyubiquitination and, thereby, NF-κB activation [11,12]. A recent report has shown that p62 is negatively implicated in Toll-like receptor 4 (TLR4)-mediated signaling through inhibition of TRAF6-evolutionarily conserved signaling intermediate in Toll pathways (ECSIT) association and the ubiquitination of ECSIT by / TRAF6 [13]. Importantly, p62− − KO mice exhibited a higher mortality rate following LPS challenge [13], suggesting that p62 might negatively regulate TLR4-mediated signaling for the activation of NF-κB. TLRs act as the first line of host defense against microbial infections and play pivotal roles in the initiation of innate immunity and the induction of adaptive immune responses by recognizing distinct pathogen-associated molecular patterns (PAMPs) [14–16]. The downstream signaling cascades of TLRs are essentially mediated by several key molecules and these include myeloid differentiation primary response 88 (MyD88), TRAF6, TAK1, mitogen-activated protein kinase kinase kinase 7 (MAP3K7), and IκB kinase (IKK) complex [14,15]. Among them, TRAF6 as an E3 ubiquitin ligase and a scaffold protein plays a key role in the TLR4-mediated activation of NF-κB[17,18]. Interestingly, recent reports have demonstrated that TLR4 and TLR3 signals induce autophagy activation, and promote migration and invasion of lung cancer cells through TRAF6 ubiquitination and MAP3K7 activation [19]. Upon TLR4 stimulation, TRAF6 promotes the K63-linked ubiquitination of BECN1 to induce TLR4-mediated autophagy [19–23]. In addition, TLR4 signaling promotes proliferation of A549 lung cancer cells through PTGS2/COX-2 (prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase)) and epidermal growth factor receptor (EGFR) activation [19]. These results strongly indicate that TLR4-induced autophagy activation and cancer cell progression might be critically linked to the TRAF6-BECN1 signaling axis. TLR4-mediated signaling induces the activation of NF-κB and autophagy induction via TRAF6-ECSIT signaling axis and TRAF6-BECN1 signaling axis, respectively [20–26]. Since p62 negatively regulates TLR4-mediated signals through the inhibition of TRAF6-ECSIT signaling [13], we therefore investigated whether p62 is functionally implicated in the TRAF6-BECN1 signaling axis, thereby regulating autophagy activation, and cancer cell migration and invasion induced by TLR4. We found that p62 interrupted the association of the TRAF6-BECN1 complex, and that inhibited BECN1 ubiquitination, leading to inhibition of autophagy activation induced by TLR4. Interestingly, p62-deficient cancer cells, p62-knockdown (p62KD) SK-HEP-1, p62KD MDA-MB-231, and p62-knockout (p62KO) A549 cells, exhibited increases in autophagic activation, and cancer cell migration/invasion induced by TLR4 stimulation. Taken together, these results suggest that p62 negatively regulates autophagy activation, thereby functionally affecting cancer cell migration and invasion induced by TLR4.

2. Materials and Methods

2.1. Cells Human embryonic kidney (HEK) 293T cells were obtained from the American Type Culture Collection (Ca# CRL-11268, ATCC, Manassas, VA, USA). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Ca#11965092, Thermo Fisher Scientific, Waltham, MA, USA). THP-1 cells, human monocytic cells, were purchased from ATCC (Ca# TIB-202), and cultured in RPMI (Roswell Park Memorial Institute) 1640 medium (Ca#11875093, Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS; Fisher Scientific HyClone, Ca#11306060), 2 mM L-glutamine (GIBCO, Ca#A2916801), 100 units/mL penicillin (GIBCO, Ca#15140122), 100 µg/mL streptomycin (GIBCO, Ca#15140122), and 5 10 5 M β-mercaptoethanol (GIBCO, Ca#21985023). Human hepatic adenocarcinoma cell line × − SK-HEP-1 (ATCC, HTB-52), human breast adenocarcinoma cell line MDA-MB-231 (ATCC, HTB-26), and human lung cancer cell line A549 (ATCC, CCL-185) were purchased from ATCC, and cultured in DMEM or RPMI contained with 10% FBS. Cells 2020, 9, 1142 3 of 14

2.2. Generation of p62-Knockdown Cell Line Lentivirus containing small hairpin RNA (shRNA) targeting human SQSTM1 (p62, Ca# sc-29679-V) and control shRNA lentivirus (Ca# sc-108080) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cells were cultured in wells of a 24-well plate (2 104 cells per well), and infected × with lentivirus, according to the manufacturer’s protocol. Control (Ctrl) THP-1, Ctrl SK-HEP-1, Ctrl MDA-MB-231 cells, p62 knockdown (p62KD) THP-1, p62KD SK-HEP-1, and p62KD MDA-MB-231 cells were cultured in puromycin-containing (4–8 µg/mL) medium, and selected as previously described [21].

2.3. Generation of p62-Knockout Cell Line with CRISPR/Cas9 Guide RNA sequences for CRISPR/Cas9 were designed at the CRISPR design web site (http://crispr.mit.edu/), provided by the Feng Zhang Lab. Insert oligonucleotides for human SQSTM1 (p62) gRNA were 50-CACCGTGGCTCCGGAAGGTGAAACACGG-30/30-CACCGAGGCC TTCCACTTTGTCAAA-50. The p62 guide RNA targets exon 2 of p62 gene. The complementary oligonucleotides for guide RNAs (gRNAs) were annealed, and cloned into lenti CRISPR v2 vector (Addgene plasmid, Ca#52961). Lenti CRISPR v2/gRNA was transfected into A549 cells by using Lipofectamine 2000, according to the manufacturer’s instructions. After two days, cells were treated with puromycin (2 µg/mL) and cultured for three days. Colonies were isolated after two weeks, and the p62 expression in the cells was confirmed by using western blot.

2.4. Antibodies and Reagents Anti-p62 antibody (Ca# ab91526) was purchased from Abcam (Cambridge, MA, USA), anti-Myc (Ca# 2276) antibody was purchased from Cell Signaling Technology (Danvers, MA, USA), and anti-Flag (Ca# F3165) and anti-HA (Ca# H3663) antibodies were purchased from Sigma-Aldrich (St Louis, MO, USA). Lipopolysaccharide (LPS, Ca#L2887), 3-methyladenine (3-MA, Ca# M9281), chloroquine (CQ, Ca# C6628), dimethyl sulfoxide (DMSO, Ca# 472301), puromycin (Ca# P9620), paraformaldehyde (Ca# P6148), Triton X-100 (Ca# T9284), gentamicin (Ca# G1272), deoxycholate (Ca# D6750), and Dulbecco’s phosphate-buffered saline (DPBS, Ca# D8537) were purchased from Sigma-Aldrich (St Louis, MO, USA). Lipofectamine 2000 (Ca# 11668-019) was purchased from Thermo Scientific (Rockford, IL, USA).

2.5. Plasmid Constructs Flag-tagged TRAF6 (Ca# 21624), Flag-tagged BECN1 (Ca# 24388), and HA-tagged p62 (Ca# 28027) were purchased from Addgene (Cambridge, MA, USA). Myc-tagged ECSIT, HA-tagged Ub, and Flag-tagged ECSIT were obtained from Dr. Jae-Hyuck Shim (University of Massachusetts Medical School, USA). The constructs coding for full-length p62 with the Flag tag or Myc tag were cloned into the pCMV-3Tag-6 vector (Agilent Technologies) or pCMV-3Tag-7 vector, respectively, using HA-tagged p62 plasmid as a template. The constructs coding for full-length BECN1 with the Myc tag were cloned into the pCMV-3Tag-7 vector, using Flag-tagged BECN1 plasmid as a template. Flag-tagged TRAF6 truncated mutants and Myc-tagged BECN1 truncated mutants were generated as previously described [21,22].

2.6. Western Blotting Analysis and Immunoprecipitation (IP) Assays Western blotting analysis and IP assays were carried out as previously described [13,20–23]. HEK293T cells were transfected with mock vector as control vector, Myc-tagged p62, or Flag-tagged TRAF6, and mock vector, Flag-tagged p62, and Myc-tagged BECN1 using Lipofectamine 2000. At 38 h after transfection, transfected cells were harvested, and cell lysates were immunoprecipitated with anti-Flag antibody. HEK293T cells were transfected with mock vector, Myc-tagged p62, or Flag-tagged TRAF6 wild type (WT) and Flag-tagged TRAF6 truncated mutants using Lipofectamine 2000. At 38 h after transfection, transfected cells were harvested, and cell lysates were immunoprecipitated with Cells 2020, 9, 1142 4 of 14 anti-Myc antibody. HEK293T cells were transfected with mock vector, Flag-tagged p62, or Myc-tagged BECN1 WT and Myc-tagged BECN1 truncated mutants using Lipofectamine 2000. At 38 h after transfection, transfected cells were harvested, and IP assay was performed with anti-Myc antibody. IP complexes were separated by SDS-PAGE (6–10%), and immune-probed with antibodies specific for anti-Myc or anti-Flag. HEK293T cells were transfected with mock vector, Flag-tagged TRAF6, and Myc-tagged BECN1, along with different concentrations of Myc-tagged p62, using Lipofectamine 2000. At 38 h after transfection, transfected cells were harvested, and IP assay was performed with with anti-Flag antibody. Immunoprecipitated complexes were separated by 6–10% SDS-PAGE, and probed with anti-Flag, anti-p62, or anti-BECN1 antibody. For ubiquitination assay, HEK293T cells were transfected with mock vector, Myc-tagged BECN1, Flag-tagged TRAF6, and HA-tagged Ub, along with different concentrations of Flag-tagged p62, using Lipofectamine 2000. At 38 h after transfection, transfected cells were harvested, and cell lysates were immunoprecipitated with anti-Myc antibody. Immunoprecipitated complexes were separated by 6–10% SDS-PAGE, and probed with anti-Myc, anti-HA, anti-p62, or anti-TRAF6 antibody. Control (Ctrl) THP-1 and p62KD THP-1 cells, Ctrl SK-HEP-1 and p62KD SK-HEP-1 cells, Ctrl MDA-MB-231 and p62KD MDA-MB-231 cells, or Ctrl A549 and p62KO A549 cells were treated with or without vehicle, 3MA (5mM), or CQ (10 µM), in the presence or absence of LPS (10 µg/mL), for 6 h. Whole cell lysates were immunoblotted with anti-LC3A/B antibody and anti-GAPDH as a loading control.

2.7. Wound-Healing and Transwell Migration Assay A wound-healing assay was carried out as previously described [21,22]. Ctrl SK-HEP-1 and p62KD SK-HEP-1 cells, Ctrl MDA-MB-231 and p62KD MDA-MB-231 cells, or Ctrl A549 and p62KO A549 cells were cultured in 12-well plates, and inoculated to confluence. Cell monolayers were gently scratched by using a sterile yellow Gilson-pipette tip to make a wide gap (approximately 400 µm). Cells were washed with culture medium, and floating cells and debris were removed from plates. Cells were treated with vehicle (DMSO, <0.2% in DMEM culture medium), 3-MA (5 mM), or CQ (10 µM) in the presence or absence of LPS (10 µg/mL), and images were captured after different times as indicated in each experiment. Transwell inserts (8µm pore; Corning, 3422) were sited into wells for cell migration assay. 5 104 cells per well were suspended in culture medium (DMEM) including × vehicle, 3-MA (5 mM), or CQ (10 µM) in the presence or absence of LPS (10 µg/mL), and placed into the top chambers of the 24-transwell plates. Culture medium, DMEM contained 10% FBS, was added to the bottom chambers. After an overnight incubation, the non-migrated cells to be remained in the top chamber were removed. The migrated cells to be existed in the bottom chamber were fixed. To visualize the nuclei, cells were stained by using crystal violet. All experiments were performed in triplicate. The experiments were repeated twice times.

2.8. Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR) Control (Ctrl) and p62KO A549 cells were treated with or without 10 µg/mL LPS for 6 h. Total RNA was extracted from cells using an RNA isolation kit (A&A Biotechnology, Gdynia, Poland) according to the manufacturer’s protocol. cDNA was obtained by RT using a amfiRivert II cDNA Synthesis Master Mix (genDEPOT, R550), according to the manufacturer’s protocol. Primers for hIL-6 (PPH 00560C), hMMP2 (PPH 00151B), and hCCL2 (PPH 00192F) were purchased from Qiagen, Inc. (Chatsworth, CA, USA). Fluorescence detection was performed using the ABI PRISM 7700 Sequence Detector (PerkinElmer; Applied Biosystems; Thermo Fisher Scientific, Inc.). The mRNA expressions were calculated and normalized to the level of GAPDH.

2.9. Statistical Analysis In vitro data are expressed as mean SEM of triplicate samples. Statistical significance of experiments ± was analyzed by using ANOVA or Student’s t-test using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). Cells 2020, 9, x FOR PEER REVIEW 5 of 14 Cells 2020, 9, 1142 5 of 14 3. Results 3. Results 3.1. P62 Interacts with TRAF6 and BECN1 3.1. P62 Interacts with TRAF6 and BECN1 It has been reported that p62 plays an inhibitory role in TLR4 signaling through interrupting the associationIt has of been TRAF6 reported with ECSIT, that p62 eventually plays an leading inhibitory to inhibition role in TLR4 of NF signaling-κB activation through [13]. interrupting TRAF6 is anthe essential association regulator of TRAF6 for the withinduction ECSIT, of eventuallyNF-κB and autophagy leading to in inhibition TLR4 signaling of NF- κ[14B– activation18]. Based [on13 ]. theseTRAF6 earlier is an reports, essential we regulator asked whether for the p62 induction is involved of NF- inκ TLR4B and-induced autophagy activation in TLR4 of signaling autophagy. [14– To18 ]. investigateBased on thesethis, we earlier first reports,examined we the asked molecular whether association p62 is involved between inp62 TLR4-induced and autophagy activation regulator of ,autophagy. suchTo as investigate TRAF6 and this, BECN1, we first in examinedTLR4 signaling. the molecular Myc-tagged association p62 or between Flag-tagged p62 p62 and expressingautophagy vector regulator were proteins,transfected such into as HEK293T TRAF6 and cells BECN1, along with in TLR4 Flag-tagged signaling. TRAF6 Myc-tagged or Myc-tagged p62 or BECN1Flag-tagged expressing p62 expressing vector, vector and werethen transfected immunoprecipitation into HEK293T (IP) cells was along performed with Flag-tagged with an TRAF6ti-Flag or antibody.Myc-tagged Consistent BECN1 expressingwith a previous vector, report and then [13], immunoprecipitation Flag-tagged TRAF6 (IP) precipitated was performed with withMyc anti-Flag-tagged p62antibody. (Supplementary Consistent Materials with a previous Figure S1A, report lane [13 4).], Flag-tagged Additionally, TRAF6 Flag-tagged precipitated p62 precipitated with Myc-tagged with Mycp62-tagged (Supplementary BECN1 (Figure Materials 1A, Figurelane 4). S1A, To determine lane 4). Additionally, the specificFlag-tagged binding site p62 of p62 precipitated to TRAF6 with or BECN1,Myc-tagged truncated BECN1 mutants (Figure1 A, of lane TRAF6 4). To or determine BECN1 werethe specific generated, binding and site IP of p62 assay to TRAF6 was performed or BECN1, betwtruncatedeen p62 mutants and these of TRAF6 truncated or BECN1 proteins were (Supplementary generated, and IPMaterials assay was Figure performed S2A,B between). As shown p62 andin Supplementarythese truncated proteinsMaterials (Supplementary Figure S3, Flag Materials-tagged Figure TRAF6 S2A,B). wild type As shown (WT) in and Supplementary Flag-tagged Materials TRAF6 truncatedFigure S3, mutants Flag-tagged were significantly TRAF6 wild precipitated type (WT) andwith Flag-tagged Myc-tagged TRAF6 p62 (Supple truncatedmentary mutants Materials were Figuresignificantly S3A, laneprecipitated 6-8), suggesting with Myc-tagged that p62 p62 interacts (Supplementary with the Materials TRAF-C Figure domain S3A, of lane TRAF6 6-8), (Supplementarysuggesting that p62Materials interacts Figure with theS3B). TRAF-C The results domain were of TRAF6 consistent (Supplementary with a previous Materials report Figure [13]. S3B).As well,The resultsMyc-tagged were BECN1 consistent WT with and aMyc previous-taggedreport BECN1 [13 1].-269 As tr well,uncated Myc-tagged mutant were BECN1 precipitated WT and withMyc-tagged Flag-tagged BECN1 p62 1-269(Figure truncated 1B, lane mutant 6 and 7), were whereas precipitated no significant with Flag-tagged interaction p62 could (Figure be observed1B, lane 6 withand Myc 7), whereas-tagged no BECN1 significant 1-127 interaction truncations could (Figure be observed 1B, lane with8), indicating Myc-tagged that BECN1 p62 interacts 1-127 truncations with the coiled(Figure-coil1B, domain lane 8), of indicating BECN1 (Figure that p62 1C). interacts with the coiled-coil domain of BECN1 (Figure1C).

Figure 1. p62 interacts with BECN1 proteins. (A) HEK293T cells were transfected with mock control Figure 1. p62 interacts with BECN1 proteins. (A) HEK293T cells were transfected with mock control vector, Flag-tagged p62, or Myc-tagged BECN1, as indicated. Transfected cells were harvested, and cell vector, Flag-tagged p62, or Myc-tagged BECN1, as indicated. Transfected cells were harvested, and lysates were immunoprecipitated with anti-Flag antibody and probed with anti-Myc or anti-Flag cell lysates were immunoprecipitated with anti-Flag antibody and probed with anti-Myc or anti-Flag antibody. (B) HEK293T cells were transfected with mock vector, Flag-tagged p62, or Myc-tagged BECN1 antibody. (B) HEK293T cells were transfected with mock vector, Flag-tagged p62, or Myc-tagged WT and Myc-tagged BECN1 truncated mutants. Transfected cells were harvested, and cell lysates BECN1 WT and Myc-tagged BECN1 truncated mutants. Transfected cells were harvested, and cell were immunoprecipitated with anti-Myc antibody. Immunoprecipitated complexes were separated by lysates were immunoprecipitated with anti-Myc antibody. Immunoprecipitated complexes were SDS-PAGE, and probed with anti-Myc or anti-Flag antibody. (C) A schematic view of the molecular separated by SDS-PAGE, and probed with anti-Myc or anti-Flag antibody. (C) A schematic view of interaction between BECN1 and p62. the molecular interaction between BECN1 and p62.

Cells 2020, 9, 1142 6 of 14 Cells 2020, 9, x FOR PEER REVIEW 6 of 14

3.2.3.2. p62 p62 Interrupts Interrupts the the Associat Associationion of of BECN1 BECN1-TRAF6-TRAF6 and and Inhibits Inhibits the the Ubiquitination Ubiquitination of of BECN1 BECN1 Induced Induced by TRAF6by TRAF6 AA previous previous study study showed showed that that TRAF6 TRAF6 interacts interacts with with the the BECN1 BECN1 and and induces induces the the ubiquitination ubiquitination ofof BECN1, BECN1, leading leading to to induction induction of of autophagy autophagy [21]. [21]. Consistently, Consistently, Flag Flag-tagged-tagged TRAF6 TRAF6 was was significantly significantly precipitatedprecipitated with with Myc Myc-tagged-tagged BECN1 BECN1 (Supplementary (Supplementary Materials Materials Figure FigureS4A). Importantly, S4A). Importantly, TRAF6 interactedTRAF6 interacted with thewith coiled the-coil coiled-coil domain domainof BECN1 of [21], BECN1 as depicted [21], as depicted in Supplementary in Supplementary Materials Materials Figure S4B.Figure As S4B.shown As in shown Figure in 1B Figure and1 1C,B,C, p62 p62 interacted interacted with with the the coiled coiled-coil-coil domain domain of of BECN1, BECN1, which which was was thethe same same binding binding domain domain as as TRAF6 TRAF6 [21]. [21]. Therefore, Therefore, we we raised raised the the possibility possibility that that p62 p62 affects affects the the molecularmolecular association association of of TRAF6 TRAF6-BECN1,-BECN1, thereby thereby inhibiting inhibiting BECN1 BECN1 ubiquitination, ubiquitination, as as depicted depicted in in FigureFigure 2A.2A. To examine examine this this possibility, possibility, we we performed performed a a competitivecompetitive binding binding assay with different concentrationsconcentrations of of p62. p62. Flag Flag-tagged-tagged TRAF6 TRAF6 and and Myc Myc-tagged-tagged BECN1 BECN1 vectors vectors were were transfected transfected into into HEK293THEK293T cells cells along along with with different different concentrations concentrations of of Myc Myc-tagged-tagged p62 p62 vectors, vectors, as as indic indicatedated Figure Figure 2B.2B. IPIP was was then then performed performed with with anti-Flag anti-Flag antibodies antibodies in cell in cell lysates. lysates. Correlating Correlating to increases to increases of Myc-tagged of Myc- taggedp62 vector, p62 the vector, interactions the interactions between Flag-tagged between Flag TRAF6-tagged and TRAF6 Myc-tagged and Myc BECN1-tagged were BECN1 significantly were significantlyattenuated (Figureattenuated2B, lane2-4;(Figure 2B, IB: lane2 BECN1-4; IB: in IPBECN with1 in Flag-tagged IP with Flag TRAF6),-tagged suggestingTRAF6), suggesting that p62 thatinterrupts p62 interrupts the molecular the molecular association association between between TRAF6 TRAF6 and BECN1. and BECN1.

FigFigureure 2. 2. p62p62 interrupts interrupts the the association association of ofTNF TNF (Tumor (Tumor necrosis necrosis factor factor)) receptor receptor-associated-associated factor factor 6 (TRAF66 (TRAF6)-BECN1)-BECN1 complex complex and and inhibits inhibits the ubiquitination the ubiquitination of BECN1 of BECN1.. (A) TRAF6 (A) TRAF6 and p62 and interact p62 interact with thewith coiled the-coil coiled-coil domain domain of BECN1. of BECN1.(B) HEK293T (B) cells HEK293T were tran cellssfected were with transfected mock vector, with mockFlag-tagged vector, TRAF6,Flag-tagged and Myc TRAF6,-tagged and BECN1, Myc-tagged along BECN1, with different along withconcentrations different concentrations of Myc-tagged of p62, Myc-tagged as indicated. p62, Trasansfected indicated. cells Transfected were harvested, cells were and harvested, cell lysates and were cell lysates immunoprecipitated were immunoprecipitated with anti-Flag with antibody anti-Flag andantibody probed and with probed anti-Flag, with anti anti-Flag,-p62, or anti-p62, anti-BECN1 or anti-BECN1 antibody. (C antibody.) HEK293T (C cells) HEK293T were tran cellssfected were withtransfected mock vector, with mock Myc- vector,tagged Myc-tagged BECN1, Flag BECN1,-tagged Flag-tagged TRAF6, and TRAF6, HA-tagged and HA-tagged Ub, along with Ub, along different with concentrationsdifferent concentrations of Flag-tagged of Flag-tagged p62, as indicated. p62, as indicated. Transfected Transfected cells were cells harvested, were harvested, and cell and lysates cell lysates were immunoprecipitated with anti-Myc antibody and probed with anti-Myc, anti-HA, anti-p62, were immunoprecipitated with anti-Myc antibody and probed with anti-Myc, anti-HA, anti-p62, or or anti-TRAF6 antibody. (D) A schematic model for how p62 interrupts the association of TRAF6-BECN1 anti-TRAF6 antibody. (D) A schematic model for how p62 interrupts the association of TRAF6-BECN1 complex and inhibits the ubiquitination of BECN1. TRAF6 interacts with the coiled-coil domain of complex and inhibits the ubiquitination of BECN1. TRAF6 interacts with the coiled-coil domain of BECN1 and induces the ubiquitination of BECN1, leading to autophagy activation. Simultaneously, BECN1 and induces the ubiquitination of BECN1, leading to autophagy activation. Simultaneously, p62 can interact with the coiled-coil domain of BECN1, and that inhibits the interaction of TRAF6 to p62 can interact with the coiled-coil domain of BECN1, and that inhibits the interaction of TRAF6 to BECN1 and the ubiquitination of BECN1. BECN1 and the ubiquitination of BECN1. Since TRAF6 interacted with BECN1 and induced its ubiquitination [19–22], we examined whether Since TRAF6 interacted with BECN1 and induced its ubiquitination [19–22], we examined inhibiting the interaction between TRAF6 and BECN1 by p62 affects BECN1 ubiquitination. To do that, whether inhibiting the interaction between TRAF6 and BECN1 by p62 affects BECN1 ubiquitination. Myc-tagged BECN1, Flag-tagged TRAF6, and HA-tagged Ub vectors were transfected into HEK293T To do that, Myc-tagged BECN1, Flag-tagged TRAF6, and HA-tagged Ub vectors were transfected cells along with different concentrations of Flag-tagged p62, and then IP was performed with anti-Myc into HEK293T cells along with different concentrations of Flag-tagged p62, and then IP was antibodies. The marginal ubiquitination of BECN1 could be seen in the absence of Flag-tagged TRAF6 performed with anti-Myc antibodies. The marginal ubiquitination of BECN1 could be seen in the (Figure2C, lane 2), whereas a marked increase could be seen in the presence of Flag-tagged TRAF6 absence of Flag-tagged TRAF6 (Figure 2C, lane 2), whereas a marked increase could be seen in the

Cells 2020, 9, 1142 7 of 14

(Figure2C, lane 3). Based on increasing Flag-tagged p62 expression, the ubiquitination of BECN1 was gradually attenuated (Figure2C, lane 4-6). These results suggest that p62 interrupts the molecular interaction between TRAF6 and BECN1, and that in turn inhibits the ubiquitination of BECN1 induced by TRAF6, as depicted in Figure2D. Studies have shown that the TRAF6-induced ubiquitination of BECN1 plays a key role in TLR-induced autophagy activation, thereby functionally implicating cancer progression and migration [19,21]. Wefound that p62 interrupted the association between BECN1 and TRAF6, and inhibited the ubiquitination of BECN1 (Figure2B,C). Therefore, we explored the functional role of p62 in autophagy induction, cancer progression, and migration, all of which are regulated by TLR4 signaling [19,21], as depicted in Figure2D.

3.3. p62-Knockdown THP-1 (p62KD THP-1), SK-HEP-1 (p62KD SK-HEP-1), and MDA-MB 231 (p62KD MDA-MB-231) Cells Exhibit Elevated Autophagy Activation Induced by TLR4 Stimulation To investigate the functional role of p62 in autophagy activation induced by TLR4 stimulation, we generated p62-knockdown THP-1 (p62KD THP-1 cells) by using the lentivirus containing shRNA targeted to p62, as described in Materials and Methods. The efficiency of p62 knockdown was significant compared to control cells (Figure3A, lane 1 versus lane 2). Control (Ctrl) THP-1 and p62KD THP-1 cells were treated with or without LPS and an autophagy inhibitor, chloroquine (CQ), as indicated in Figure3B, and autophagy activation was assessed by western blotting with anti-LC3 antibody. Upon LPS stimulation, the level of LC3-II increased in both cell lines, (Figure3B, lane 1 versus lane 2 in Ctrl THP-1 and lane 4 versus lane 5 in p62KD THP-1), but the increase in LC3-II was significantly higher in p62KD THP-1 cells than in Ctrl THP-1 cells (Figure3B, lane 2 versus lane 5; Figure3C, open bars versus closed bars in LPS). As expected, CQ treatment induced the marked accumulation of LC3-II in both cell lines (Figure3B, lane 3 and lane 6; Figure3C, open bars versus closed bars in LPS plus CQ), suggesting that p62 negatively regulates autophagy activation induced by TLR4 stimulation. To investigate further the role of p62 in activating autophagy, we generated p62-knockdowns in two cancer cell lines, p62KD SK-HEP-1 and p62KD MDA-MB 231 cells, as described in Materials and Methods. The efficacy of p62 knockdown in SK-HEP-1 and MDA-MB-231 cells was significant as compared to control cells (Figure3D in SK-HEP-1 and 3G in MDA-MB-231 cells, lane 1 versus lane 2). In similar fashion to p62KD THP-1 cells (Figure3B,C), the levels of LC3-II were significantly enhanced in p62KD SK-HEP-1 and p62KD MDA-MB-231 cells in the presence of LPS, as compared to their controls (Figure3E,F in SK-HEP-1 lane 2 versus lane 5 and open bars versus closed bars in LPS treated; Figure3H,I in MDA-MB-231, lane 2 versus lane 5 and open bars versus closed bars in LPS treated). These results suggest that p62 negatively regulates autophagy activation induced by TLR4 stimulation, presumably by the inhibition of the ubiquitination of BECN1 as demonstrated in Figure2D.

3.4. p62-Deficient Cancer Cells Exhibit Increased Cancer Cell Migration and Invasion, Induced by TLR4 Stimulation Having shown that p62 negatively regulated autophagy activation, we asked whether the inhibitory effect was functionally associated with cancer cell migration and invasion. To do that, migration and invasion assays were performed in p62KD SK-HEP-1 and p62KD MDA-MB-231 cells. Ctrl SK-HEP-1 and p62KD SK-HEP-1 were treated with vehicle, LPS, LPS plus a 3-methyladenine (3-MA) autophagy inhibitor, and LPS plus a CQ autophagy inhibitor, and then wound healing assay was performed. Based on LPS treatment, cancer cell migratory behavior was significantly higher in p62KD SK-HEP-1 than the Ctrl SK-HEP-1 cells in a time dependent manner (Figure4A,B, Ctrl versus p62KD SK-HEP-1 in LPS treatment). These results were consistently observed in Ctrl and p62KD MDA-MB-231 cells (Figure4C,D, Ctrl versus p62 KD MDA-MB 231 in LPS treatment). As expected, marked attenuations could be seen in co-treatments with 3-MA or CQ (Figure4A,B, Ctrl versus p62 KD SK-HEP-1 in LPS plus 3-MA or CQ: Figure4C,D, Ctrl versus p62 KD MDA-MB-231 in LPS plus 3-MA or Cells 2020, 9, x FOR PEER REVIEW 7 of 14 presence of Flag-tagged TRAF6 (Figure 2C, lane 3). Based on increasing Flag-tagged p62 expression, the ubiquitination of BECN1 was gradually attenuated (Figure 2C, lane 4-6). These results suggest that p62 interrupts the molecular interaction between TRAF6 and BECN1, and that in turn inhibits the ubiquitination of BECN1 induced by TRAF6, as depicted in Figure 2D. Studies have shown that the TRAF6-induced ubiquitination of BECN1 plays a key role in TLR- induced autophagy activation, thereby functionally implicating cancer progression and migration [19,21]. We found that p62 interrupted the association between BECN1 and TRAF6, and inhibited the ubiquitination of BECN1 (Figure 2B,C). Therefore, we explored the functional role of p62 in autophagy induction, cancer progression, and migration, all of which are regulated by TLR4 signaling [19,21], as depicted in Figure 2D.

3.3. p62-Knockdown THP-1 (p62KD THP-1), SK-HEP-1 (p62KD SK-HEP-1), and MDA-MB 231 (p62KD MDA-MB-231) Cells Exhibit Elevated Autophagy Activation Induced by TLR4 Stimulation To investigate the functional role of p62 in autophagy activation induced by TLR4 stimulation, we generated p62-knockdown THP-1 (p62KD THP-1 cells) by using the lentivirus containing shRNA targeted to p62, as described in Materials and Methods. The efficiency of p62 knockdown was significant compared to control cells (Figure 3A, lane 1 versus lane 2). Control (Ctrl) THP-1 and p62KD THP-1 cells were treated with or without LPS and an autophagy inhibitor, chloroquine (CQ), as Cells 2020, 9, 1142 8 of 14 indicated in Figure 3B, and autophagy activation was assessed by western blotting with anti-LC3 antibody. Upon LPS stimulation, the level of LC3-II increased in both cell lines, (Figure 3B, lane 1 versusCQ). We lane next 2 in assessed Ctrl THP invasiveness-1 and lane following 4 versus TLR4lane 5 stimulation. in p62KD THP Ctrl-1), SK-HEP-1 but the increase and p62 inKD LC3SK-HEP-1-II was significantlywere treated withhigher vehicle, in p62 LPS,KD THP LPS-1 plus cells 3-MA, than andin Ctrl LPS THP plus-1 CQ, cells and (Figure then transwell3B, lane 2 migration versus lane assay 5; Figurewas performed. 3C, open Similarbars versus to the closed wound bars healing in LPS). assay, As progressive expected, invasivenessCQ treatment was induced significantly the marked higher accumulationin p62KD SK-HEP-1 of LC3 than-II in inboth Ctrl cell SK-HEP-1 lines (Figure in the 3B, presence lane 3 and of LPS lane (Figure 6; Figure5A,B, 3C, Ctrl open versus bars p62versusKD closedSK-HEP-1 bars in in LPS LPS treatment). plus CQ), suggesting Consistent that results p62 were negatively observed regulates in Ctrl autophagy and p62KD MDA-MB-231activation induced cells by(Figure TLR45C,D, stimulation. Ctrl versus p62 KD MDA-MB-231 in LPS treatment).

KD KD KD FigFigureure 3. 3. p62p62-deficient-deficient cells, cells, p62 p62KD THPTHP-1,-1, p62KD p62 SK-HEPSK-HEP-1,-1, and p62 andKD p62MDA-MBMDA-MB-231-231 cells, exhibit cells, KD enhancedexhibit enhanced autophagy autophagy activation activation in response in response to TLR4 to TLR4 stimulation stimulation.. (A) p62 (AKD) p62THP-1 THP-1 cells were cells generated,were generated, and the and knockdown the knockdown efficacy effi ofcacy p62 of was p62 confirmed was confirmed with withanti-p62 anti-p62 antibody antibody.. (B,C) ( BCtrl,C) and Ctrl KD p62andKD p62 THP-THP-11 cells cellswere weretreated treated with withor without or without vehicle vehicle or CQ or CQ(10 μM), (10 µ M),in the in thepresence presence or absence or absence of of LPS (10 µg/mL), for 6 h. Whole cell lysates were immunoblotted with anti-LC3A/B antibody and anti-GAPDH antibody as a loading control (B). The LC3II levels were analyzed with Image J program (C). Data shown are averages from a minimum of 3 independent experiments ( SEM). *, p < 0.05. ± (D) p62KD SK-HEP-1 cells were generated, and the knockdown efficacy of p62 was confirmed with anti-p62 antibody. (E,F) Ctrl and p62KD SK-HEP-1 were treated with or without vehicle or CQ, in the presence or absence of LPS. Whole cell lysates were immunoblotted with anti-LC3A/B and anti-GAPDH antibodies (E). The LC3II levels were analyzed with Image J program (F). Data shown are averages from a minimum of 3 independent experiments ( SEM). *, p < 0.05 and **, p < 0.01. (G) p62KD ± MDA-MB-231 cells were generated, and the knockdown efficacy of p62 was confirmed with anti-p62 antibody. (H,I) Ctrl and p62KD MDA-MB-231 were treated with or without vehicle or CQ, in the presence or absence of LPS. Whole cell lysates were immunoblotted with anti-LC3A/B and anti-GAPDH antibodies (H). The LC3II levels were analyzed with Image J program (I). Data shown are averages from a minimum of 3 independent experiments ( SEM). * p < 0.05. ± To verify the above results, we generated p62-knockout (p62KO) A549 cells using Crispr cas9 (Figure6A). Upon TLR4 stimulation, the levels of LC3-II were significantly increased in both Ctrl A459 and p62KO A549 cells (Figure6B, lane 1 versus lane 2 in Ctrl A549 and lane 4 versus lane 5 in p62 KO A549 cells). Moreover, the LC3-II ratio relative to control was significantly higher in p62KO A549 cells than in Ctrl A549 (Figure6C, closed bar versus open bar in LPS). As expected, treatments of autophagy inhibitor CQ or 3-MA induced increased or decreased LC3-II levels, respectively (Figure6B, lane 3 and lane 4 in Ctrl; lane 6 and 7 in p62KO A549). Consistent with results of p62KD SK-HEP-1 and p62KD Cells 2020, 9, 1142 9 of 14

MDA-MB 231, the cell migration and invasion activities induced by LPS were markedly enhanced in p62KO A549 cells compared to controls, whereas significant attenuations were observed in co-treatments with 3-MA or CQ (Figure6D,E, Ctrl versus p62 KO A549 in LPS treatment; Figure6F,G, Ctrl versus p62KO A549 in LPS treatment). It has been previously reported that TLR4-induced autophagy activation promoted migration and invasion of lung cancer by induction of chemokines and immunosuppressive factors including CCL2, CCL20, IL-6, VEGFA, and MMP2 [19,27,28]. Therefore, we evaluated the production of IL-6, MMP2, and CCL2 in Ctrl and p62KO A549 cells in the presence or absence of LPS stimulation. Consistent with the earlier report, the levels of IL-6 mRNA, MMP2 mRNA, CCL2 mRNA were significantly elevated in Ctrl A549 cells treated with LPS (Figure6H, without LPS versus with LPS in Ctrl A549). Interestingly, these levels were markedly higher in p62KO A549 cells than in Ctrl A549 cells under LPS stimulation (Figure6H, Ctrl versus p62 KO A549 with LPS). Collectively these results suggest that p62 negatively regulates autophagy activation, cancer cell migration and invasion Cellsinduced 2020, 9 by, x FOR TLR4 PEER stimulation. REVIEW 9 of 14

Figure 4. KD KD Figure 4. p62p62KD SKSK-HEP-1-HEP-1 and and p62 p62KD MDAMDA-MB-231-MB-231 cells cells exhibit exhibit increased increased cell cell migration migration in in response response to TLR4 stimulation. (A,B) Ctrl and p62KD SK-HEP-1 cells were seeded into 12-well cell culture to TLR4 stimulation. (A,B) Ctrl and p62KD SK-HEP-1 cells were seeded into 12-well cell culture plates. plates. Confluent monolayers were scraped with a sterile yellow Gilson-pipette tip, and the wound Confluent monolayers were scraped with a sterile yellow Gilson-pipette tip, and the wound was then was then treated with vehicle (DMSO, <0.2% in culture medium), LPS (10 µg/mL), 3-MA (5 mM) treated with vehicle (DMSO, <0.2% in culture medium), LPS (10 μg/mL), 3-MA (5 mM) plus LPS (10 plus LPS (10 µg/mL), and CQ (10 µM) plus LPS (10 µg/mL) for different time periods, as indicated. μg/mL), and CQ (10 μM) plus LPS (10 μg/mL) for different time periods, as indicated. A A representative experiment is shown (A). The residual gap between the migrating cells from the representative experiment is shown (A). The residual gap between the migrating cells from the opposing wound edge was expressed as a percentage of the initial scraped area ( SEM, n = 3) (B). opposing wound edge was expressed as a percentage of the initial scraped area (± ±SEM, n = 3) (B). *, *, p < 0.05 and **, p < 0.01.(C,D) Ctrl and p62KD MDA-MB-231 were seeded into 12-well cell culture p< 0.05 and **, p < 0.01. (C,D) Ctrl and p62KD MDA-MB-231 were seeded into 12-well cell culture plates. plates. Confluent monolayers were scraped with a sterile yellow Gilson-pipette tip, and the wound Confluent monolayers were scraped with a sterile yellow Gilson-pipette tip, and the wound was then was then treated with vehicle (DMSO, <0.2% in culture medium), LPS (10 µg/mL), 3-MA (5 mM) treated with vehicle (DMSO, <0.2% in culture medium), LPS (10 μg/mL), 3-MA (5 mM) plus LPS (10 plus LPS (10 µg/mL), and CQ (10 µM) plus LPS (10 µg/mL) for different time periods, as indicated. μg/mL), and CQ (10 μM) plus LPS (10 μg/mL) for different time periods, as indicated. A A representative experiment is shown (C). The residual gap between the migrating cells from the representative experiment is shown (C). The residual gap between the migrating cells from the opposing wound edge was expressed as a percentage of the initial scraped area ( SEM, n = 3) (D). opposing wound edge was expressed as a percentage of the initial scraped area (± ±SEM, n = 3) (D). * * p < 0.05 and ** p < 0.01. p < 0.05 and ** p < 0.01.

To verify the above results, we generated p62-knockout (p62KO) A549 cells using Crispr cas9 (Figure 6A). Upon TLR4 stimulation, the levels of LC3-II were significantly increased in both Ctrl A459 and p62KO A549 cells (Figure 6B, lane 1 versus lane 2 in Ctrl A549 and lane 4 versus lane 5 in p62KO A549 cells). Moreover, the LC3-II ratio relative to control was significantly higher in p62KO A549 cells than in Ctrl A549 (Figure 6C, closed bar versus open bar in LPS). As expected, treatments of autophagy inhibitor CQ or 3-MA induced increased or decreased LC3-II levels, respectively (Figure 6B, lane 3 and lane 4 in Ctrl; lane 6 and 7 in p62KO A549). Consistent with results of p62KD SK-HEP-1 and p62KD MDA-MB 231, the cell migration and invasion activities induced by LPS were markedly enhanced in p62KO A549 cells compared to controls, whereas significant attenuations were observed in co-treatments with 3-MA or CQ (Figure 6D,E, Ctrl versus p62KO A549 in LPS treatment; Figure 6F,G, Ctrl versus p62KO A549 in LPS treatment). It has been previously reported that TLR4-induced autophagy activation promoted migration and invasion of lung cancer by induction of chemokines and immunosuppressive factors including CCL2, CCL20, IL-6, VEGFA, and MMP2 [19,27,28]. Therefore, we evaluated the production of IL-6, MMP2, and CCL2 in Ctrl and p62KO A549 cells in the presence or absence of LPS stimulation. Consistent with the earlier report, the levels of IL-6 mRNA, MMP2 mRNA, CCL2 mRNA were significantly elevated in Ctrl A549 cells treated with LPS (Figure 6H, without LPS versus with LPS in Ctrl A549). Interestingly, these levels were markedly higher in p62KO A549 cells than in Ctrl A549 cells under LPS stimulation (Figure 6H, Ctrl versus p62KO A549 with LPS). Collectively these results suggest that p62 negatively regulates autophagy activation, cancer cell migration and invasion induced by TLR4 stimulation.

Cells 2020, 9, x FOR PEER REVIEW 10 of 14 Cells 2020, 9, 1142 10 of 14 Cells 2020, 9, x FOR PEER REVIEW 10 of 14

Figure 5. p62KD SK-HEP-1 and p62KD MDA-MB-231 cells exhibit increased invasiveness in response KD KDKD FigFigureure 5. p62 SKSK-HEP-1-HEP-1 and p62p62 MDAMDA-MB-231KD -MB-231 cellscells exhibitexhibit increased increased invasiveness invasiveness in in response response to to TLR4 stimulation. (A,B) Ctrl and p62KD SK-HEP-1 cells were suspended in DMEM culture medium TLR4 stimulation. (A,B) Ctrl and p62 KDSK-HEP-1 cells were suspended in DMEM culture medium toincluding TLR4 stimulation. vehicle, LPS (A ,(10B) Ctrlμg/mL) and, p623-MA SK(5 -mM)HEP -plus1 cells LPS were (10 suspendedμg/mL), and in CQDMEM (10 μM) culture plus medium LPS (10 including vehicle, LPS (10 µg/mL), 3-MA (5 mM) plus LPS (10 µg/mL), and CQ (10 µM) plus LPS includingμg/mL). Cells vehicle, were LPS placed (10 μ intog/mL) the, 3 top-MA chambers (5 mM) plus of 24 LPS-transwell (10 μg /mL)plates, and and CQ incubated (10 μM) for plus overnight. LPS (10 (10 µg/mL). Cells were placed into the top chambers of 24-transwell plates and incubated for overnight. μFixedg/mL) cells. Cells were were stained placed by into using the topcrystal chambers violet (ofA ).24 Numbers-transwell of plates migrated and incubatedcells were for coun overnight.ted, and Fixed cells were stained by using crystal violet (A). Numbers of migrated cells were counted, and results Fixed cells were stained by using crystal violet (A). Numbers of migratedKD cells were counted, and results are represented as mean ± SEM (B).* p< 0.05. (C,D) CtrlKD and p62 MDA-MB-231 cells were are represented as mean SEM (B). * p < 0.05. (C,D) Ctrl and p62 MDA-MB-231KD cells were suspended resultssuspended are represented in culture medium ±as mean of ± RPMI SEM including(B).* p< 0.05. vehicle, (C,D LPS) Ctrl (10 and μg /mL)p62 , 3 MDA-MA (5-MB mM)-231 plus cells LPS were (10 in culture medium of RPMI including vehicle, LPS (10 µg/mL), 3-MA (5 mM) plus LPS (10 µg/mL), suspendedμg/mL), and in cultureCQ (10 mediumμM) plus of LPS RPMI (10 includingμg/mL). Cells vehicle, were LPS placed (10 μ ginto/mL) top, 3 -chambersMA (5 mM) of plus24-transwell LPS (10 and CQ (10 µM) plus LPS (10 µg/mL). Cells were placed into top chambers of 24-transwell plates and μplatesg/mL) and, and further CQ (10 incubated μM) plus for LPS overnight. (10 μg/mL) Fixed. Cells cells were were placed stained into with top crystal chambers violet of ( C24).- transwellNumbers further incubated for overnight. Fixed cells were stained with crystal violet (C). Numbers of migrated platesof migrated and further cells were incubated counted, for andovernight. results Fixed are represented cells were stainedas mean with ± SEM crystal (D).* violet p< 0.05. (C ). Numbers cells were counted, and results are represented as mean SEM (D). * p < 0.05. of migrated cells were counted, and results are represented± as mean ± SEM (D).* p< 0.05.

KO FigureFigure 6. 6. p62p62KO A549 cells exhibited increased autophagy activation, activation, migration,migration, andand invasion invasion in in KO KO Figresponseresponseure 6. totop62 TLR4TLR4 A549 stimulation.stimulation. cells exhibited ((AA)) p62p62 KO increased A549 cells autophagy were generated, activation, and migration the knockout, and eefficacyffi invasioncacy ofof p62p62 in response to TLR4 stimulation. (A) p62KO A549 cells were generated, and the knockout efficacy of p62

Cells 2020, 9, 1142 11 of 14

was confirmed with anti-p62 antibody. (B,C) Ctrl and p62KO A549 cells were treated with or without vehicle, CQ (10 µM), or 3-MA (5 mM), or, in the presence or absence of LPS (10 µg/mL), for 6 h. Whole cell lysates were immunoblotted with anti-LC3A/B antibody and anti-GAPDH antibody as a loading control (B). The LC3II levels were analyzed with Image J program (right, histogram) (C). Data shown are averages from a minimum of 3 independent experiments ( SEM). * p < 0.05 and ** p < 0.01.(D,E) Ctrl ± and p62KO A549 cells were seeded into 12-well cell culture plates. Confluent monolayers were scraped with a sterile yellow Gilson-pipette tip to make wounds, and then incubated with vehicle (DMSO, <0.2% in culture medium), LPS (10 µg/mL), 3-MA (5 mM) plus LPS (10 µg/mL), and CQ (10 µM) plus LPS (10 µg/mL) for different time periods. A representative experiment is represented (D). The residual gap between the migrating cells from the opposing wound edge was represented as a percentage of the initial scraped area ( SEM, n = 3) (E). ** p < 0.01. (F,G) Ctrl and p62KO A549 cells were suspended ± in RPMI medium including vehicle, LPS (10 µg/mL), 3-MA (5 mM) plus LPS (10 µg/mL), and CQ (10 µM) plus LPS (10 µg/mL), and placed to the top chambers of 24-transwell plates. After an overnight incubation, cells were fixed and stained with crystal violet (F). Number of migrating cell were counted, and results are presented as mean SEM of 3 independent experiments (G). * p < 0.05 and ** p < 0.01. ± (H) Control (Ctrl) and p62KO A549 cells were treated with or without 10 µg/mL LPS for 6 h. Total RNA was extracted, cDNA was obtained, as described in materials and methods, and RT-qPCR analysis performed with specific primers, such as hIL-6, hMMP2, and hCCL2. * p < 0.05.

4. Discussion In this study, we demonstrate that p62, which is a known negative regulator for the activation of autophagy by TLR4 signaling, inhibits the ubiquitination of BECN1 mediated by TRAF6. We demonstrate this occurs through an interruption to the molecular association between TRAF6 and BECN1 and, thereby, involves cancer cell migration and invasion, activities that are facilitated by autophagy, as depicted in Figure7. Through the biochemical studies, we found that p62 competitively interacted with the coiled-coil domain of BECN1 affecting binding to TRAF6, and that induced an attenuation of BECN1 ubiquitination. Levels of LC3-II revealed increased autophagy induction in the presence of TLR4 stimulation by p62KD THP-1 and p62KD cancer cells. More importantly, these p62-deficient cancer cells, including p62KO A549 cells, showed enhanced migration and invasion in response to TLR4 stimulation. Taken together, these results suggest that p62 is negatively implicated in the activation of autophagy by TLR4 signaling, thereby demonstrating an involvement in cancer cell invasiveness facilitated by autophagy induction. Accumulating evidence suggests that TLR signaling might be critical for induction and activation of autophagy, thereby facilitating cancer cell migration and invasion [19,21]. Among signal cascades, TRAF6 plays a pivotal role in NF-κB activation and autophagy induction through the activation of TLR downstream molecules and the ubiquitination of BECN1, respectively [19–26]. A previous study demonstrated that p62 attenuated the ubiquitination of ECSIT, which is known as one of the regulators in TLR4-mediated signaling for NF-κB activation, induced by TRAF6 [13]. Interestingly, we found in the current study that p62 interacted with BECN1, as well as TRAF6. The molecular associations between p62 and BECN1 or TRAF6 revealed that p62 interacted with the coiled-coil domain of BECN1, which also surprisingly served as the interaction site for TRAF6 [21], possibly explaining the inhibitory mechanism of p62 in the association of TRAF6-BECN1. p62 interference in TRAF6-BECN1 complex formation, eventually induced the attenuation of BECN1 ubiquitination by TRAF6. Another important finding in the current study was that p62 regulated cancer cell invasiveness was facilitated by TLR4 stimulation through autophagy activation. Although there is some controversy over whether autophagy activation is either positively or negatively involved in cancer metastasis [29–33], recent studies have demonstrated that TLRs induced cancer cell migration and invasion through facilitating autophagy induction, which was mechanistically associated with the TRAF6-BECN1 signaling axis [19,21]. Consistent with these reports, we found that p62-deficient cancer cells, exhibited elevated autophagy activation, cancer cell migration, and invasiveness in response to TLR4 stimulation. Moreover, TCGA (The Cancer Genome Atlas) data revealed that the expression of p62 was significantly lower in primary Cells 2020, 9, x FOR PEER REVIEW 11 of 14

was confirmed with anti-p62 antibody. (B,C) Ctrl and p62KO A549 cells were treated with or without vehicle, CQ (10 μM), or 3-MA (5 mM), or, in the presence or absence of LPS (10 μg/mL), for 6 h. Whole cell lysates were immunoblotted with anti-LC3A/B antibody and anti-GAPDH antibody as a loading control (B). The LC3II levels were analyzed with Image J program (right, histogram) (C). Data shown are averages from a minimum of 3 independent experiments (± SEM). * p < 0.05 and ** p < 0.01. (D,E) Ctrl and p62KO A549 cells were seeded into 12-well cell culture plates. Confluent monolayers were scraped with a sterile yellow Gilson-pipette tip to make wounds, and then incubated with vehicle (DMSO, <0.2% in culture medium), LPS (10 μg/mL), 3-MA (5 mM) plus LPS (10 μg/mL), and CQ (10 μM) plus LPS (10 μg/mL) for different time periods. A representative experiment is represented (D). The residual gap between the migrating cells from the opposing wound edge was represented as a percentage of the initial scraped area (± SEM, n = 3) (E). ** p < 0.01. (F,G) Ctrl and p62KO A549 cells were suspended in RPMI medium including vehicle, LPS (10 μg/mL), 3-MA (5 mM) plus LPS (10 μg/mL), and CQ (10 μM) plus LPS (10 μg/mL), and placed to the top chambers of 24-transwell plates. After an overnight incubation, cells were fixed and stained with crystal violet (F). Number of migrating cell were counted, and results are presented as mean ± SEM of 3 independent experiments Cells 2020(G, 9).*, 1142 p < 0.05 and ** p < 0.01. (H) Control (Ctrl) and p62KO A549 cells were treated with or without 10 12 of 14 μg/mL LPS for 6 h. Total RNA was extracted, cDNA was obtained, as described in materials and methods, and RT-qPCR analysis performed with specific primers, such as hIL-6, hMMP2, and hCCL2. tumors,* p such < 0.05. as colorectal cancer, breast cancer, prostate adenocarcinoma/prostate cancer, and bladder cancer/bladder urothelial carcinoma, than those of normal primary cells (Supplementary Materials Figure4. Discussion S5A–D). In addition, metastatic tumors, such as clear cell renal cell carcinoma, kidney cancer,I andn this colorectal study, we cancer, demonstrate showed that greater p62, which downregulation is a known ofnegative p62, compared regulator tofor primary the activation tumors (Supplementaryof autophagy by Materials TLR4 signaling, Figure S6A,B). inhibits These the ubiquitinationresults strongly of suggest BECN1 that mediated p62 may by beTRAF6. negatively We associateddemonstrate with this autophagy occurs through activation an in interruption cancer cells to induced the molecular by TLR4 association stimulation, between and thereby TRAF6 be and a key regulatorBECN1 ofand cancer, thereby cell metastasis., involves cancer cell migration and invasion, activities that are facilitated by autophagy,In summary, as we depicted propose in a Figure molecular 7. Throughmechanism the for, biochemical and functional studies, effects we of, found p62 in autophagy that p62 activationcompetitively and cancer interacted progressions with the coiled induced-coilby domain TLR4 of stimulation, BECN1 affecting as depicted binding in to Figure TRAF6,7. Thereand that are someinduced controversies an attenuation related of to BECN1 p62 and ubiquitination. its involvement Levels in of inflammatory LC3-II revealed versus increased anti-inflammatory autophagy responsesinduction [9 – in12 ]. the Considering presence of p62 TLR4 is a versatile stimulation adaptor by p62 proteinKD THP with-1 multiple and p62KD cellular cancer functions cells. More [1–8 ], theimportantly, outstanding these issues p62 need-deficient to be ca clarifiedncer cells, in including the near p62 future.KO A549 Our cells, results showed contribute enhanced to migration a growing understandingand invasion of in the response multi-functional to TLR4 stimulation. role of p62 Taken in autophagy together, and these cancer results progression. suggest that This p62 new is understandingnegatively implicated may prove in the useful activation for the of autophagy development by TLR4 of promising signaling, therapeutic thereby demonstrating approaches an for treatmentinvolvement of inflammatory in cancer cell diseases invasiveness and cancersfacilitated in certainby autophagy pathological induction. conditions.

FigureFigure 7. 7.p62 p62 is is negatively negatively involved involved in in autophagy autophagy activation, activation, and and cancer cancer cell cell migration migration and and invasion invasion in responsein response to TLR4 to TLR4 stimulation. stimulation. Engagement Engagement of TLR4 of TLR4 ligand ligand leads leads to the to association the association of TRAF6-BECN1 of TRAF6- complex,BECN1 andcomplex, that induces and that the induces ubiquitination the ubiquitination of BECN1. of BECN1 BECN1. ubiquitination BECN1 ubiqu inducesitination the induces activation the of autophagy, thereby potentially regulating cancer progression, via migration and invasion, as depicted in the upper panel. However, the interaction between p62 and BECN1 inhibits the association of TRAF6 to BECN1, and that inhibits the ubiquitination of BECN1, leading to inhibitions of autophagy activation, and cancer cell migration and invasion, as depicted in the lower panel.

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4409/9/5/1142/s1, Figure S1: TRAF6 interacts with p62.; Figure S2: Generations of TRAF6 or BECN1 truncated mutants.; Figure S3: p62 interacts with the TRAF-C domain of TRAF6.; Figure S4: TRAF6 interacts with the coiled-coil domain of BECN1.; Figure S5: The association between SQSTM1/p62 expression and primary tumors.; Figure S6: The association between SQSTM1/p62 expression and tumor metastasis. Author Contributions: Conceptualization, K.-Y.L.; validation, K.-Y.L., M.-J.K., and Y.M.; formal analysis, M.-J.K., and Y.M.; investigation, M.-J.K., Y.M., J.S.L., and J.S.I.; data curation, K.-Y.L., M.-J.K., and Y.M.; writing-original draft preparation, K.-Y.L., and J.S.L.; writing- review and editing, K.-Y.L., and J.S.L.; visualization, K.-Y.L., M.-J.K., Y.M., and J.S.I.; supervision, K.-Y.L.; project administration, K.-Y.L., and M.-J.K.; funding acquisition, K.-Y.L., and J.S.L. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by a grant of the National Research Foundation of Korea (NRF), funded by the Korean Government (NRF-2018R1D1A1B07042470) and the Korean Government (MSIP) (2016R1A5A2945889). Acknowledgments: We would like to thank Hyehwa Forum members for their helpful discussions. Conflicts of Interest: The authors declare no conflict of interest. Cells 2020, 9, 1142 13 of 14

References

1. Geetha, T.; Wooten, M.W. Structure and functional properties of the ubiquitin binding protein p62. FEBS Lett. 2002, 512, 19–24. [CrossRef] 2. Seibenhener, M.L.; Babu, J.R.; Geetha, T.; Wong, H.C.; Krishna, N.R.; Wooten, M.W. Sequestosome 1/p62 is a polyubiquitin chain binding protein involved in ubiquitin proteasome degradation. Mol. Cell. Biol. 2004, 24, 8055–8068. [CrossRef] 3. Pugsley, H.R. Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry. J. Vis. Exp. 2017, 125, e55637. [CrossRef] 4. Jiang, P.; Mizushima, N. LC3- and p62-based biochemical methods for the analysis of autophagy progression in mammalian cells. Methods 2015, 75, 13–18. [CrossRef] 5. Ichimura, Y.; Waguri, S.; Sou, Y.S.; Kageyama, S.; Hasegawa, J.; Ishimura, R.; Saito, T.; Yang, Y.; Kouno, T.; Fukutomi, T.; et al. Phosphorylation of p62 activates the Keap1-Nrf2 pathway during selective autophagy. Mol. Cell 2013, 51, 618–631. [CrossRef] 6. Durán, A.; Serrano, M.; Leitges, M.; Flores, J.M.; Picard, S.; Brown, J.P.; Moscat, J.; Diaz-Meco, M.T. The atypical PKC-interacting protein p62 is an important mediator of RANK-activated osteoclastogenesis. Dev. Cell 2004, 6, 303–309. [CrossRef] 7. Puls, A.; Schmidt, S.; Grawe, F.; Stabel, S. Interaction of protein kinase C zeta with ZIP, a novel protein kinase C-binding protein. Proc. Natl. Acad. Sci. USA 1997, 94, 6191–6196. [CrossRef] 8. Sanz, L.; Diaz-Meco, M.T.; Nakano, H.; Moscat, J. The atypical PKC-interacting protein p62 channels NF-kappaB activation by the IL-1-TRAF6 pathway. EMBO J. 2000, 19, 1576–1586. [CrossRef] 9. Into, T.; Inomata, M.; Niida, S.; Murakami, Y.; Shibata, K. Regulation of MyD88 aggregation and the MyD88-dependent signaling pathway by sequestosome 1 and histone deacetylase 6. J. Biol. Chem. 2010, 285, 35759–35769. [CrossRef] 10. Kim, J.Y.; Ozato, K. The sequestosome 1/p62 attenuates cytokine in activated macrophages by inhibiting IFN regulatory factor 8 and TNF receptor-associated factor 6/NF-kappaB activity. J. Immunol. 2009, 182, 2131–2140. [CrossRef] 11. Duran, A.; Linares, J.F.; Galvez, A.S.; Wikenheiser, K.; Flores, J.M.; Diaz-Meco, M.T.; Moscat, J. The signaling adaptor p62 is an important NF-kappaB mediator in tumorigenesis. Cancer Cell 2008, 13, 343–354. [CrossRef] [PubMed] 12. Wooten, M.W.; Seibenhener, M.L.; Mamidipudi, V.; Diaz-Meco, M.T.; Barker, P.A.; Moscat, J. The atypical protein kinase C-interacting protein p62 is a scaffold for NF-kappaB activation by nerve growth factor. J. Biol. Chem. 2001, 276, 7709–7712. [CrossRef][PubMed] 13. Kim, M.J.; Min, Y.; Kwon, J.; Son, J.; Im, J.S.; Shin, J.; Lee, K.Y. p62 Negatively Regulates TLR4 Signaling via Functional Regulation of the TRAF6-ECSIT Complex. Immune Netw. 2019, 19, e16. [CrossRef][PubMed] 14. Akira, S.; Takeda, K.; Kaisho, T. Toll-like receptors: Critical proteins linking innate and acquired immunity. Nat. Immunol. 2001, 2, 675–680. [CrossRef] 15. Kopp, E.B.; Medzhitov, R. The Toll-receptor family and control of innate immunity. Curr. Opin. Immunol. 1999, 11, 13–18. [CrossRef] 16. Ninomiya-Tsuji, J.; Kishimoto, K.; Hiyama, A.; Inoue, J.; Cao, Z.; Matsumoto, K. The kinase TAK1 can activate the NIK-I kappaB as well as the MAP kinase cascade in the IL-1 signalling pathway. Nature 1999, 398, 252–256. [CrossRef] 17. Kawai, T.; Akira, S. TLR signaling. Cell Death Differ. 2006, 13, 816–825. [CrossRef] 18. Kawasaki, T.; Kawai, T. Toll-like receptor signaling pathways. Front. Immunol. 2014, 5, 461. [CrossRef] 19. Zhan, Z.; Xie, X.; Cao, H.; Zhou, X.; Zhang, X.D.; Fan, H.; Liu, Z. Autophagy facilitates TLR4- and TLR3-triggered migration and invasion of lung cancer cells through the promotion of TRAF6 ubiquitination. Autophagy 2014, 10, 257–268. [CrossRef] 20. Kim, M.J.; Min, Y.; Shim, J.H.; Chun, E.; Lee, K.Y. CRBN Is a Negative Regulator of Bactericidal Activity and Autophagy Activation Through Inhibiting the Ubiquitination of ECSIT and BECN1. Front Immunol. 2019, 10, 2203. [CrossRef] 21. Min, Y.; Kim, M.J.; Lee, S.; Chun, E.; Lee, K.Y. Inhibition of TRAF6 ubiquitin-ligase activity by PRDX1 leads to inhibition of NFKB activation and autophagy activation. Autophagy 2018, 14, 1347–1358. [CrossRef] Cells 2020, 9, 1142 14 of 14

22. Min, Y.; Lee, S.; Kim, M.J.; Chun, E.; Lee, K.Y. Ubiquitin-Specific Protease 14 Negatively Regulates Toll-Like Receptor 4-Mediated Signaling and Autophagy Induction by Inhibiting Ubiquitination of TAK1-Binding Protein 2 and Beclin 1. Front Immunol. 2017, 8, 1827. [CrossRef] 23. Min, Y.; Wi, S.M.; Kang, J.A.; Yang, T.; Park, C.S.; Park, S.G.; Chung, S.; Shim, J.H.; Chun, E.; Lee, K.Y. Cereblon negatively regulates TLR4 signaling through the attenuation of ubiquitination of TRAF6. Cell Death Dis. 2016, 7, e2313. [CrossRef] 24. Xiao, C.; Shim, J.H.; Klüppel, M.; Zhang, S.S.; Dong, C.; Flavell, R.A.; Fu, X.Y.; Wrana, J.L.; Hogan, B.L.; Ghosh, S. Ecsit is required for Bmp signaling and mesoderm formation during mouse embryogenesis. Dev. 2003, 17, 2933–2949. [CrossRef] 25. Wi, S.M.; Moon, G.; Kim, J.; Kim, S.T.; Shim, J.H.; Chun, E.; Lee, K.Y. TAK1-ECSIT-TRAF6 complex plays a key role in the TLR4 signal to activate NF-κB. J. Biol. Chem. 2014, 289, 35205–35214. [CrossRef] 26. Mi, W.S.; Park, J.; Shim, J.H.; Chun, E.; Lee, K.Y. Ubiquitination of ECSIT is crucial for the activation of p65/p50 NF-κBs in Toll-like receptor 4 signaling. Mol. Biol. Cell 2015, 26, 151–160. [CrossRef] 27. Kelly, M.G.; Alvero, A.B.; Chen, R.; Silasi, D.A.; Abrahams, V.M.; Chan, S.; Visintin, I.; Rutherford, T.; Mor, G. TLR-4 signaling promotes tumor growth and paclitaxel chemoresistance in ovarian cancer. Cancer Res. 2006, 66, 3859–3868. [CrossRef] 28. Hattar, K.; Savai, R.; Subtil, F.S.; Wilhelm, J.; Schmall, A.; Lang, D.S.; Goldmann, T.; Eul, B.; Dahlem, G.; Fink, L.; et al. Endotoxin induces proliferation of NSCLC in vitro and in vivo: Role of COX-2 and EGFR activation. Cancer Immunol. Immunother. 2013, 62, 309–320. [CrossRef] 29. Peng, Y.F.; Shi, Y.H.; Ding, Z.B.; Ke, A.W.; Gu, C.Y.; Hui, B.; Zhou, J.; Qiu, S.J.; Dai, Z.; Fan, J. Autophagy inhibition suppresses pulmonary metastasis of HCC in mice via impairing anoikis resistance and colonization of HCC cells. Autophagy 2013, 9, 2056–2068. [CrossRef] 30. Peng, Y.F.; Shi, Y.H.; Shen, Y.H.; Ding, Z.B.; Ke, A.W.; Zhou, J.; Qiu, S.J.; Fan, J. Promoting colonization in metastatic HCC cells by modulation of autophagy. PLoS ONE 2013, 8, e74407. [CrossRef] 31. Galavotti, S.; Bartesaghi, S.; Faccenda, D.; Shaked-Rabi, M.; Sanzone, S.; McEvoy, A.; Dinsdale, D.; Condorelli, F.; Brandner, S.; Campanella, M.; et al. The autophagy-associated factors DRAM1 and p62 regulate cell migration and invasion in glioblastoma stem cells. Oncogene 2013, 32, 699–712. [CrossRef] 32. Kiyono, K.; Suzuki, H.I.; Matsuyama, H.; Morishita, Y.; Komuro, A.; Kano, M.R.; Sugimoto, K.; Miyazono, K. Autophagy is activated by TGF-beta and potentiates TGF-beta-mediated growth inhibition in human hepatocellular carcinoma cells. Cancer Res. 2009, 69, 8844–8852. [CrossRef] 33. Li, J.; Yang, B.; Zhou, Q.; Wu, Y.; Shang, D.; Guo, Y.; Song, Z.; Zheng, Q.; Xiong, J. Autophagy promotes hepatocellular carcinoma cell invasion through activation of epithelial-mesenchymal transition. Carcinogenesis 2013, 34, 1343–1351. [CrossRef]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).