www.impactjournals.com/oncotarget/ Oncotarget, Vol. 7, No. 32 Research Paper PATZ1 induces PP4R2 to form a negative feedback loop on IKK/ NF-κB signaling in lung cancer Ming-Yi Ho1, Chi-Ming Liang1, Shu-Mei Liang1,2 1Genomics Research Center, Academia Sinica, Taipei 11529, Taiwan, ROC 2Agricultural Biotechnology Research Center, Academia Sinica, Taipei 11529, Taiwan, ROC Correspondence to: Chi-Ming Liang, email: [email protected] Shu-Mei Liang, email: [email protected] Keywords: epithelial-mesenchymal transition (EMT), IκB kinase (IKK), migration/invasion/metastasis, PATZ1, protein phosphatase 4 (PP4) Received: February 29, 2016 Accepted: June 17, 2016 Published: July 6, 2016 ABSTRACT Activation of IKK enhances NF-κB signaling to facilitate cancer cell migration, invasion and metastasis. Here, we uncover the existence of a negative feedback loop of IKK. The transcription factor PATZ1 induces protein phosphatase-4 (PP4) regulatory subunit 2 (PP4R2) in an IKK-dependent manner. PP4R2 enhances the binding of PP4 to phosphorylated IKK to inactivate IKK/NF-κB signaling during sustained stimulation by cellular stimuli such as growth factors and inflammatory mediators. Matched pair studies reveal that primary lung cancers express more PATZ1 and PP4R2 than lymph node metastases in patients. Ectopic PATZ1 decreases invasion/colonization of lung cancers and prolongs the survival of xenograft mice. These effects of PATZ1 are reversed by downregulating PP4R2. Our results suggest that PATZ1 and PP4R2 provide negative feedback on IKK/NF-κB signaling to prevent cancer cells from over-stimulation from cellular stimuli; a decline in PATZ1 and PP4R2 is functionally associated with cancer migration/invasion and agents enhancing PATZ1 and PP4R2 are worth exploring to prevent invasion/metastasis of lung cancers. INTRODUCTION [5]. The PGE2-mediated increase of phospho-AktS473 dissipates within 2 h and reappears late after 12 h [6]. Growth factors, inflammatory cytokines, pathogens The resurgence of phospho-AktS473 is due to migration- and cellular stresses enhance NF-κB activity that regulates inducing factor 7 (MIG-7)-mediated inactivation of many target genes resulting in a variety of cellular protein phosphatase 2A (PP2A) [6]. It is not clear, responses [1]. NF-κB activity is inhibited by the binding of however, whether this resurgence of phospho-AktS473 is IκB which masks the nuclear localization sequence of NF- also accompanied by an increase in phosphorylated IKK κB to modulate its regulation of downstream genes [2]. and NF-κB p65. The inhibitory effect of IκB on NF-κB is in turn suppressed Phosphorylation and dephosphorylation of signal by the phosphorylated IκB kinase (phospho-IKK) that mediators or effectors such as Akt, IKK and NF-κB phosphorylates IκB to increase polyubiquitination and by kinases and phosphatases serve as a reversible degradation of IκB [3]. Constitutive, aberrant elevation mechanism to switch on and off stimuli-mediated cellular of active NF-κB is associated with chronic inflammatory processes like metabolism, apoptosis, proliferation and diseases and many types of cancer [4]. migration [5, 7]. The majority of oncogenes encode Stimulation of lung cancer cells with growth factors kinases to facilitate phosphorylation of signal mediators, and inflammatoryRETRACTED mediators such as PGE2 increased the whereas phosphatases such as PP2A primarily suppress level of PIP3 to reach a plateau at 1 h and then declined phosphorylation of signal mediators or effectors to serve [5]. This transient increase in PIP3 was accompanied by an as cancer suppressors [8-10]. To monitor cellular activities, increase in phosphorylated Akt and prohibitin (phospho- it is important to examine not only how quickly a signal AktS473 and phospho-PHBT258) resulting in transient mediator or effector such as Akt, IKK or NF-κB p65 has upregulation of phosphorylation of IKK and NF-κB p65 been phosphorylated and activated but also how long they remain in the activated (phosphorylated) state. www.impactjournals.com/oncotarget 52255 Oncotarget In this study, we found that even though phospho- PP4R2/PP4C suppresses IKK phosphorylation AktS473 in lung cancer cells reappeared 12 h after PGE2 or growth factor stimulation, this resurgence of Signal mediators or effectors are known to be phospho-AktS473 was not accompanied by an increase in dephosphorylated by phosphatases [7]. We thus co- S176/180 phosphorylated IKK and NF-κB p65. IKK and NF-κB p65 immunoprecipitated phospho-IKKα/β with several were sustained in the dephosphorylated state due to PP4. potential phosphatase candidates to examine whether We also found that the increase in the effect of PP4 was any of the phosphatases can bind and dephosphorylate S176/180 primarily due to the increase in PP4 regulatory subunit phospho-IKKα/β during the late phase of PGE2 2 (PP4R2) that enhances the association of phospho- or growth factor stimulation. Our results showed that IKKα/βS176/180 with PP4 catalytic unit (PP4C) leading to during the late phase, PP4C but not PP1, PP2A or PP5 S176/180 dephosphorylation of phospho-IKKα/βS176/180. Additional co-immunoprecipitated with phospho-IKKα/β studies revealed that the induction of PP4R2 was primarily (Figure 1C). As PP4C is known to be activated by due to a transcription factor, namely POZ/BTB and AT- PP4R1, PP4R2, PP4R3α/β or PP4R4, that may form a hook-containing zinc finger protein 1 variant 4 (PATZ1) holoenzyme with PP4C to increase its binding to the target [11, 12]. The biological impact of PATZ1and PP4R2/PP4C molecules [14, 15], we also evaluated whether PP4R1, negative feedback on IKK/NF-κB was further elucidated PP4R2, PP4R3α/β or PP4R4 could co-immunoprecipitate S176/180 by upregulating and downregulating PATZ1 and PP4R2 with phospho-IKKα/β . Our results showed that to study their influence on the cellular effects of growth there was an increase in PP4R2 but not PP4R1 co- S176/180 factors, PGE2 and PIP3 and the migration/invasion and immunoprecipitated with phospho-IKKα/β during colonization/metastasis of lung cancer cells in vitro and the late phase, whereas PP4 R3α/β and PP4R4 did not S176/180 in vivo. interact with phospho-IKKα/β in lung cancer cells (Figure 1C). In addition, there was more PP4R2 but not RESULTS PP4R1 co-immunoprecipitated with PP4C (Supplementary Figure S2A). PP4R2 by itself did not dephosphorylate IKK (Supplementary Figure S2B); PP4C caused slight Homeostasis of IKK/NF-κB signaling dephosphorylation of IKK and this dephosphorylating Stimulation of lung cancer cells with PGE2 or effect of PP4C was increased dramatically in the presence growth factors (EGF, HGF, IGF-1) results in transient of PP4R2 (Supplementary Figure S2B). increase of phospho-AktS473, phospho-IKKα/βS176/180 and We then examined whether or not downregulation phospho-NF-κB p65S536 in the early phase (within 1 h) of PP4C and PP4R2 increases the level of phosphorylated [5]. As the increase of phospho-AktS473 has been shown to IKK and NF-κB. Our results revealed that selective dissipate within 2 h and reappear in the late phase (about downregulation of PP4C decreased IκBα and increased not S176/180 12-48 h later) [6], we examined whether phospho-IKKα/ only the levels of phospho-IKKα/β and phospho-NF- S536 βS176/180 and phospho-NF-κB p65S536 in lung cancer cells κB p65 but also COX-2 and Snail in the late phase of could be increased along with phospho-AktS473 during PGE2 stimulation (Supplementary Figure S3A and S3B; the late phase of PGE2 stimulation. Our results showed right panels). Interestingly, although knockdown of PP4C that neither phospho-IKKα/βS176/180 nor phospho-NF-κB downregulated cellular PP4C activity, it did not affect the p65S536 was increased during the late phase (Figure 1A). amount of PP4R2 co-immunoprecipitated with phospho- S176/180 Since phospho-IKKα/βS176/180 and phospho-NF-κB p65S536 IKKα/β (Supplementary Figure S3C; right panels). may increase cellular COX-2 and epithelial-mesenchymal Knockdown of PP4R2 decreased IκBα and enhanced S176/180 S536 transition (EMT) [13], we analyzed the levels of COX- phospho-IKKα/β , phospho-NF-κB p65 , COX-2 as 2, vimentin and E-cadherin transcription suppressors, well as Snail in the late phase of PGE2 stimulation (Figure notably Snail, ZEB-1 and Twist during the late phase. 2A and 2B; left panels). It also abolished the association of S176/180 We found that the levels of ZEB-1 reached a plateau at PP4C with phospho-IKKα/β (Figure 2C; left panels). 24-25 h and stayed at a relatively high level for a much Unlike knockdown of PP4C and PP4R2, downregulation longer time (> 17 h; Figure 1B). The level of Twist which of control siRNA (Supplementary Figure S3) or PP4R1 is known to be increased by phospho-Akt-induced MIG- (Figure 2A, 2B, 2C; right panels) did not show significant S176/180 S536 7 [6], was elevated at 27 h and remained at high level in effects on phospho-IKKα/β , phospho-NF-κB p65 , the late phase (Figure 1B). In comparison, the levels of COX-2 and Snail. COX-2 and SnailRETRACTED peaked at 24-25 h and then decreased to the basal level 11 h later (Figure 1B), suggesting that they PATZ1 increases PP4R2 expression might be IKK/NF-κB dependent. Similar results were not only found in PGE2-treated (Figure 1) but also PIP3- and To find out what factor(s) might bind to the promoter growth factor-treated lung cancer cells (Supplementary region of PP4R2 gene to enhance PP4R2 expression, Figure S1). we searched the TRANSFAC [16] and DECODE (SABiosciences, Frederick, MD, USA) databases. Both www.impactjournals.com/oncotarget 52256 Oncotarget databases indicated that PATZ1 would be the most 6 h before the increase of PP4R2 that occurred at 12 h likely candidate. Since PATZ1 is known to exist as four after PGE2 stimulation (Figure 3A and Supplementary alternatively spliced transcript variants with distinct Figure S2).