Identification of XAF1–MT2A Mutual Antagonism As a Molecular Switch in Cell-Fate Decisions Under Stressful Conditions

Identification of XAF1–MT2A mutual antagonism as a molecular switch in cell-fate decisions under stressful conditions

Cheol-Hee Shina, Min-Goo Leea, Jikhyon Hana, Seong-In Jeonga, Byung-Kyu Ryua, and Sung-Gil Chia,1

aDepartment of Life Sciences, Korea University, Seoul 02841, Korea

Edited by Carol Prives, Columbia University, New York, NY, and approved April 26, 2017 (received for review January 18, 2017)

XIAP-associated factor 1 (XAF1) is a tumor suppressor that is which in turn deregulates the apoptotic process and contributes commonly inactivated in multiple human neoplasms. However, to tumorigenesis (8). However, it was shown that XAF1 evokes −/− the molecular mechanism underlying its proapoptotic function an apoptotic effect in XIAP cells to the extent comparable in +/+ remains largely undefined. Here, we report that XAF1 induction by XIAP cells, and XIAP is not sequestered to the nucleus in heavy metals triggers an apoptotic switch of stress response by cells undergoing XAF1-induced apoptosis, indicating that destabilizing metallothionein 2A (MT2A). XAF1 directly interacts XAF1’s proapoptotic function is not solely dependent on the with MT2A and facilitates its lysosomal degradation, resulting in XIAP-interfering activity (6, 9). XAF1 was also identified as an the elevation of the free intercellular zinc level and subsequent IFN-stimulated gene that contributes to IFN-dependent sensi- activation of p53 and inactivation of XIAP. Intriguingly, XAF1 is tization of cells to tumor necrosis factor (TNF)-related apopto- activated as a unique transcription target of metal-regulatory sis inducing ligand (TRAIL)-induced apoptosis (10, 11). Recently, transcription factor-1 (MTF-1) in signaling apoptosis, and its pro- we reported that XAF1 forms a positive feedback loop with tein is destabilized via the lysosomal pathway by MTF-1–induced the p53 tumor suppressor and acts as a molecular switch in MT2A under cytostatic stress conditions, indicating the presence of p53-mediated cell-fate decisions favoring apoptosis over cell- mutual antagonism between XAF1 and MT2A. The antagonistic cycle arrest (12). In this process, XAF1 appears to promote interplay between XAF1 and MT2A acts as a key molecular switch homeodomain-interacting protein kinase 2 (HIPK2)-mediated in MTF-1–mediated cell-fate decisions and also plays an important p53 phosphorylation by interrupting the HIPK2-targeting function of Siah2 and promotes zinc finger protein 313 (ZNF313)- role in cell response to various apoptotic and survival factors. induced p21WAF1 ubiquitination (13). XAF1 was also shown to Wild-type (WT) XAF1 but not MT2A binding-deficient mutant inhibit tumor angiogenesis by suppressing VEGF expression and XAF1 increases the free intracellular zinc level and accelerates regulate the cell cycle through modulating the G2/M checkpoint WT folding of p53 and degradation of XIAP. Consistently, XAF1 p53+/+ and interaction with checkpoint kinase 1 (14, 15). evokes a more drastic apoptotic effect in versus isogenic Many heavy metals and metallic agents have biological activity p53−/− cells. Clinically, expression levels of XAF1 and MT2A are but also cause many adverse health effects, such as carcinoge- inversely correlated in primary colon tumors and multiple cancer nicity in humans (16). However, the mechanism underlying cell lines. XAF1-depleted xenograft tumors display an increased heavy-metal–induced tumorigenesis remains unclear. Metal- growth rate and a decreased apoptotic response to cytotoxic lothioneins (MTs) are low-molecular-weight (6–7 kDa), cysteine- heavy metals with strong MT2A expression. Collectively, this study rich, heavy-metal–binding proteins that play a crucial role in the uncovers an important role for XAF1–MT2A antagonism as a linch- regulation of intracellular metal metabolism and metal-induced pin to govern cell fate under various stressful conditions including stresses (17, 18). Upon heavy metals stimuli, MTs are rapidly heavy metal exposure. Significance XAF1 | metallothionein | MTF-1 | heavy metal | apoptosis MEDICAL SCIENCES Epigenetic inactivation of XAF1 tumor suppressor is frequently limination of defective or potentially dangerous cells by ap- observed in multiple human cancers. However, the mechanisms Eoptosis plays a fundamental role in the regulation of tissue underlying its function remain largely undefined. Here, we pre- homeostasis and prevention of malignant transformation and sent evidence that XAF1 plays a critical role in cell-fate decisions autoimmunity (1). X-linked inhibitor of apoptosis (XIAP)- under heavy-metal–induced stress conditions through the mu- associated factor 1 (XAF1) is a proapoptotic tumor suppressor tual antagonism with MT2A. XAF1 is activated as a transcription that is identified as a negative regulator of XIAP based on its target of MTF-1 and destabilizes MT2A through the interaction- ability to bind and interfere with the anticaspase function of directed lysosomal degradation, whereas it is destabilized by XIAP (2, 3). XAF1 expression is down-regulated in a broad MT2A under cytostatic stress conditions. XAF1-mediated MT2A range of human tumors mainly by aberrant promoter CpG site inactivation leads to elevation of free intracellular zinc level and hypermethylation, and its reduction is associated with the ad- up- and down-regulates p53 and XIAP, respectively, to promote vanced stage and high grade of many cancers (4–6). A recent apoptosis. XAF1–MT2A antagonism thus represents one critical study showed that PTEN-null mouse prostate tumors showing regulator of cell-fate decisions, suggesting that it could be an resistance to androgen-deprivation therapy have reduced levels attractive target for the therapeutic intervention of tumor of XAF1, and diminished expression of XAF1 by continuous progression. androgen deprivation therapy is positively correlated with met- astatic castrate-resistant tumor progression (7). Author contributions: C.-H.S., M.-G.L., and S.-G.C. designed research; C.-H.S., M.-G.L., J.H., XAF1 sensitizes tumor cells to the proapoptotic effects of S.-I.J., and B.-K.R. performed research; C.-H.S., M.-G.L., J.H., S.-I.J., B.-K.R., and S.-G.C. analyzed data; and C.-H.S. and S.-G.C. wrote the paper. chemotherapeutic drugs, γ-irradiation, UV, H2O2, hypoxia, and growth factor deprivation, whereas its depletion increases cel- The authors declare no conflict of interest. lular resistance to apoptotic stresses (5, 6). XAF1 was originally This article is a PNAS Direct Submission. identified as a nuclear protein that could bind and sequester 1To whom correspondence should be addressed. Email: [email protected]. XIAP protein to the nucleus (3). It was thus proposed that loss of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. XAF1 may increase the functional pool of cytoplasmic XIAP, 1073/pnas.1700861114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1700861114 PNAS | May 30, 2017 | vol. 114 | no. 22 | 5683–5688 Downloaded by guest on September 28, 2021 plays a crucial role in cell-fate decisions under various stressful A IP B C In vitro MT2A conditions, including heavy metal exposure. pull-down 1 28 50 80 106 132 301 binding -+-GST ZF1 ZF2 ZF3 ZF4 ZF5 ZF6 ZF7 WT +

IgG XAF1 MT2A --+ GST-XAF1 N1 + Results IB: XAF1 -++r-MT2A-His N2 + N3 IB: MT2A IB: His + XAF1 Directly Interacts with and Down-Regulates MT2A. In an at- GST-XAF1 N4 + XAF1 N5 - tempt to understand the mechanistic basis of XAF1 proapoptotic MT2A 7C + 6C function, we initially performed a yeast two-hybrid assay and WCL GST + Tubulin 5C - A MT2A-His identified MT2A as a XAF1-interacting protein (Fig. S1 ). The HCC1937 ZF5 - – GFP-XAF1 XAF1 MT2A interaction was confirmed by immunoprecipita- DEtion (IP) and GST pull-down assays (Fig. 1A and Fig. S1 B and 7C 6C 5C N1 N2 N3 N4 N5 WT WT C). An in vitro pull-down assay using purified GST-XAF1 and + + + + + + + + + + + + MT2A recombinant MT2A proteins revealed that XAF1 binds directly B pcDNA IB: GFP WT-XAF1 si-Cont si-XAF1 to MT2A (Fig. 1 ). Using a series of deletion mutants, we de- MT2A termined that zinc finger domain 5 (ZF5) of XAF1 is essential

IP: GFP IB XAF1 IB: MT2A C D Tubulin for the interaction (Fig. 1 and ). Moreover, the MT2A MT2A RT- MT2A protein level was markedly decreased and increased by XAF1 WCL Tubulin PCR GAPDH expression and depletion, respectively, suggesting that XAF1 U2OS F G U2OS U2OS/GFP-MT2A may down-regulate MT2A via direct interaction (Fig. 1E and Fig. -+ -WT-XAF1 --+ ZF5-XAF1 - - WT ZF5 XAF1 S1 E and F). As predicted, MT2A protein level was reduced by IB: GFP wild type (WT)-XAF1 but not affected by ΔZF5-XAF1, a mu- MT2A (Input) GFP (MT2A) tant XAF1 lacking the ZF5 domain (Fig. 1F). Consistently, an IP: MT2A immunofluorescence assay using U2OS/GFP-MT2A subline cells XAF1 (GFP) MT2A XAF1 showed a profound reduction of GFP-MT2A fluorescence by

WCL Δ G Tubulin X 200 WT-XAF1 but not by ZF5-XAF1 (Fig. 1 ). Based on these observations, we asked whether XAF1 regulates MT1A, another Fig. 1. XAF1 binds directly to MT2A. (A) Interaction of endogenously ubiquitously expressed MT isoform, and found that XAF1 also expressed XAF1 and MT2A. IB, immunoblot; WCL, whole-cell lysate. (B)In interacts with and down-regulates MT1A (Fig. S1 F–I). vitro pull-down assay showing a direct interaction between XAF1 and MT2A. (C) XAF1 constructs and its MT2A-binding status. (D) Identification of the XAF1 Promotes Lysosomal Degradation of MTs. XAF1 controls ZF5 domain as a MT2A-binding region of XAF1. (E) XAF1 down-regulation of proteasomal degradation of proteins through the interaction MT2A protein expression. (F) A crucial role for XAF1 ZF5 domain in binding with ubiquitin E3 ligases (12, 13). Meanwhile, the protein sta- and downregulating MT2A. (G) Immunofluorescence microscopic analysis bility of MTs has been known to be regulated mainly through the Δ showing no MT2A-reducing activity of ZF5-XAF1. lysosomal pathway (33). We determined whether XAF1 down- regulation of MTs is affected by the proteasome inhibitor MG132 and/or the lysosome inhibitor Leupeptin. In HCT116 cells transcriptionally activated by metal-regulatory transcription with a tetracycline-inducible system (Tet-XAF1), XAF1 down- factor-1 (MTF-1) and function in protecting cells against heavy regulation of MT2A and MT1A was blocked by Leupeptin but metals, particularly cadmium, zinc, copper, mercury, nickel, not affected by MG132 (Fig. 2A and Fig. S2A). A cycloheximide platinum, and silver primarily by acting as scavengers of toxic (CHX) chase experiment revealed that the half-life of MT2A metal ions (19, 20). MTs also have crucial roles in cell proliferation and differentiation, and their activity to bind heavy metals and detoxify free radicals can help cancer cells to survive by inhibition of apoptosis (21, 22). Moreover, MTF-1 is elevated ABHCT116 C (Tet-XAF1) 1 HCT116 (Tet-XAF1) in human breast, lung, and cervical carcinoma-derived cell lines, HCT116 (Tet-XAF1) Tet - - + + + Tet - + Tet 0.8 Tet + supporting the importance of MTs in carcinogenesis (23). - - + - MG132 Of the 11 human functional MT isoforms [MT-1 (A, B, E, F, ---+ Leupeptin 0 3 6 12 24 0 3 6 12 24 CHX (h) 0.6 MT2A G, H, M, and X), MT2A, MT-3, and MT-4], ubiquitously MT2A 0.4 XAF1 XAF1 0.2 expressed MT1 and MT2A are extensively studied for their Tubulin Tubulin MT2A protein level 0 U2OS (Tet-XAF1) 0 3 6 12 24 ability to attenuate metal-induced toxicity and as biomarkers in D E Post-CHX treatment (h) many human cancers (18, 24, 25). MT2A is the major MT iso- MT2A Lysosome Merge MKN74 0 0.5 1 2 GFP-MT2A ( g) form expressed in humans and has specific functions in regu- - MT2A (GFP) lating apoptosis, autophagy, and increasing risk of cancers (26– IB XAF1

28). MT2A is up-regulated in many types of tumor tissues and Tet Tubulin + RT- XAF1 cell lines, and its elevation is associated with adverse prognosis. PCR GAPDH H X400 1 HCC1937 MT2A induction decreases intracellular zinc availability whereas LysoTracker DAPI pcDNA HCC1937 0.8 MT2A its silencing increases free intracellular zinc concentration, thus F G HCC1937 - + + + GFP-MT2A 0.6 modulating the intracellular levels of bioavailable zinc (29). - - + - MG132 pcDNA GFP-MT2A MT2A contributes to chemotherapy resistance by direct chela- ---+ Leupeptin 0 2 4 6 8 0 2 4 6 8 CHX (h) 0.4 XAF1 XAF1 0.2

tion of zinc and platinum-containing drugs and by indirect action MT2A MT2A XAF1 protein level 0 Tubulin Tubulin 0 2 4 6 8 on p53 zinc-dependent activity. It was reported that HIPK2 de- Post-CHX treatment (h) pletion induces MT2A up-regulation and p53 misfolding, but codepletion of MT2A restores p53 activity, showing that MT2A Fig. 2. XAF1 promotes lysosomal degradation of MT2A. (A) Blockade of can modulate p53 through zinc exchange (30). Several studies XAF1-induced MT2A reduction by Leupeptin. Cells were treated with tetra- μ μ μ demonstrated an association of MT2A elevation with grade and cycline (1 g/mL) and then exposed to MG132 (10 M) or Leupeptin (10 M) for aggressiveness of tumors, including prostate cancer (27, 31, 32). 6h.(B and C) A CHX chase assay showing XAF1-mediated MT2A destabilization. Cells were treated with CHX (40 μM) for the indicated hours. Nevertheless, the molecular mechanism underlying the bi- (D) Immunofluorescence analysis showing XAF1 stimulation of MT2A lysosomal ological functions of MT2A and its regulation in the carcinogenic translocation. (E) MT2A down-regulation of XAF1 protein expression. (F) Blockade process has not been well understood. of MT2A-induced XAF1 reduction by Leupeptin. (G and H)ACHXchaseassay In this study, we present evidence that XAF1–MT2A mutual showing MT2A-mediated XAF1 destabilization (G) and comparison of XAF1 pro- antagonism via the interaction-directed lysosomal degradation tein levels in control and MT2A-overexpressed cells following CHX treatment (H).

5684 | www.pnas.org/cgi/doi/10.1073/pnas.1700861114 Shin et al. Downloaded by guest on September 28, 2021 is blocked (Fig. 3B). In MTF-1–overexpressed cells, XAF1 in- A B HCT116 2 2 2 duction suppressed MT2A protein but not mRNA induction. As si-Control si-XAF1 expected, a putative MRE was identified in the 5′ upstream re- CdCl Control ZnCl CuCl 0 0.5 1 2 0 0.5 1 2 MTF-1 ( g) + -+ -+ -+-si-Control MTF-1 gion (nucleotides −283/−277 relative to ATG) of the XAF1 gene, -+ -+ -+ -+si-MTF-1 XAF1 and the reporters comprising this MRE displayed an approxi- RT- XAF1 PCR GAPDH IB MT2A mately fivefold increase in luciferase activity in response to C. PARP MTF-1 CdCl2 and ZnCl2 whereas this responsiveness was not detected Tubulin IB XAF1 XAF1 in MTF-1–depleted cells (Fig. 3 C–E). A chromatin IP (ChIP) Tubulin RT- MT2A assay revealed that CdCl exposure induces MTF-1 binding to PCR 2 C 5’-TGCCCAG-3’ GAPDH the MRE in the XAF1 promoter, indicating that the MRE is -1537 -500 -221 +1 E HCT116 (XAF1-Pro500-Luc) occupied by endogenous MTF-1 (Fig. 3F). Expression assays MRE Luc Pro1537 6 ** using increasing doses of ZnCl2 treatment showed that XAF1 MRE Luc Pro500 5 expression is activated in response to cytotoxic (≥1 μM) but not 4 D Luc Pro221 cytostatic (0.1–0.5 μM) stress whereas MT2A is induced in re- 3 6 HCC1937 ** Control sponse to both stresses (Fig. 3G). ** 2 ** CdCl2 5 ** ZnCl 2 1 4 XAF1–MT2A Antagonism Plays a Crucial Role in Cell-Fate Decisions 0 0102550 0102550 ZnCl (M) 3 activity Relative luciferase 2 Under Metal Stress Conditions. We investigated whether the 2 G si-Control si-MTF-1 XAF1–MT2A antagonism is linked to cellular response to metal 1 HCT116 stress. In cells exposed to cytostatic doses of CdCl2 (<5 μM), 0 0.10.20.5 1 2 5 10 20 ZnCl (M) 0 2 ZnCl2 (<1 μM), and CuCl2 (<100 μM), MT2A was up-regulated

Relative luciferase activity Relative luciferase pGL3 1537 500 221 XAF1 RT- A B MT2A and XAF1 was down-regulated (Fig. 4 and ). By contrast, XAF1-Pro-Luc PCR GAPDH F HCC1937 when the cells were exposed to cytotoxic doses, XAF1 was up- C. PARP regulated and MT2A was down-regulated. The metal dose- Input IgG -MTF-1 IB Tubulin -+ -+ -+ CdCl2 (12 h) associated reduction of XAF1 and MT2A was blocked by de- XAF1 (MRE) Cytostatic Cytotoxic Cell response pletion of MT2A and XAF1, respectively, indicating that the GAPDH (1.3 - 1.7) (9.8 - 43.6) (Sub-G1 %) opposite responses of XAF1 and MT2A to metal stress results C Fig. 3. Identification of XAF1 as a direct transcriptional target of MTF-1. from their mutual antagonism (Fig. 4 ). Compared with control (A)ActivationofXAF1 mRNA expression by heavy metals. Cells were treated cells, XAF1-depleted cells manifested a substantially decreased with heavy metal (100 μM) for 24 h. (B) MTF-1 induction of apoptosis and its apoptotic response to cytotoxic metal stress, and XAF1- attenuation by XAF1 depletion. (C)AMREintheXAF1 promoter and reporter overexpressed cells showed apoptotic response to cytostatic construction for luciferase assay. (D and E) Metal activation of the XAF1 pro- metal stress (Fig. 4D and Fig. S4 A–D). ΔZF5-XAF1 failed to moter and its attenuation by MTF-1 depletion. (F) A ChIP assay showing MTF-1 evoke this effect. Either XAF1 overexpression or MT2A de- interaction with the MRE in the XAF1 promoter. (G) Activation of XAF1 mRNA pletion directed apoptotic switch of cellular response to cytostatic expression by cytotoxic but not cytostatic doses of ZnCl2. stress, and either MT2A overexpression or XAF1 depletion protected cells from cytotoxic stress (Fig. 4 C and E and Fig. S4D). Moreover, MT2A depletion-induced apoptotic sensitization was protein is shortened from 8.9 h to 3.1 h by XAF1 induction (Fig. 2 debilitated when XAF1 was codepleted, and XAF1 depletion- B and C). In U2OS/GFP-MT2A cells, GFP-MT2A fluorescence induced cell protection was abolished when MT2A was code- was decreased by XAF1, but this effect was abolished by Leupeptin pleted (Fig. S4 E–I). (Fig. S2B). Moreover, XAF1 was shown to stimulate the lysosomal translocation of MT2A and MT1A, supporting that XAF1 facilitates lysosomal degradation of MTs (Fig. 2D and Fig. S2C). In- triguingly, XAF1 was also down-regulated at the protein level by A HCC1937

MT2A and MT1A, and this effect was blocked by Leupeptin, in- CdCl2 ( M, 48 h) ZnCl2 ( M, 48 h) CuCl2 ( M, 48 h) 0 0.10.5 1 5 10 20 40 0 0.10.20.5 1 5 10 20 0 5 10 25 50100200400 dicating that both MTs promote lysosomal degradation of XAF1 MEDICAL SCIENCES (Fig. 2 E and F and Fig. S2 D and E). The half-life of XAF1 protein XAF1 F MT2A was shortened from 5.7 h to 3.3 h by overexpressed MT2A (Fig. 2 C. PARP and G). MT2A failed to decrease ΔZF5-XAF1, supporting that Tubulin MT2A destabilizes XAF1 through the interaction (Fig. S2F). We B HCC1937 C HCC1937 0.5 M 20 M ZnCl2 next assessed whether interaction of XAF1 and MT2A results in si-Control si-MT2A si-Control si-XAF1 their mutual degradation. Compared with individually transfected 0 6 12 24 48 0 6 12 24 48 (h) MT2A 0 0.1 0.5 1 0 0.10.5 1 0 5 10 20 0 5 10 20 CdCl2 (M) cells, cotransfected cells showed detectable reduction of both pro- XAF1 XAF1 MT2A teins, and this reduction of both proteins was blocked by Leupeptin C. PARP C. Casp-3 C. PARP (Fig. S2 G–I). Together, these results support the presence of Tubulin – Tubulin XAF1 MT2A mutual antagonism. CdCl (20 M, 24 h) D 2 E 0 0 1 2 WT ( g) HCT116 XAF1 Is a Unique Transcriptional Target of MTF-1 in Signaling 1.72 19.07 28.32 37.86 Sub-G1 XAF1 ( g) MT2A ( g) (%) Apoptosis. Next we examined whether XAF1 is activated in re- 0 0 0.5 1 2 0 0 0.5 1 2 0 1 1 1 1 0 20 20 20 20 CdCl2 (M) sponse to heavy metals. In multiple human cells exposed to 0 0 1 2 ZF5 ( g) XAF1 1.83 22.27 24.18 24.94 Sub-G1 MT2A CdCl2, ZnCl2, and CuCl2, XAF1 expression was markedly up- HCT116 regulated at the mRNA level, and its induction showed an in- (%) C. PARP verse correlation with MT2A at the protein level (Fig. S3 A–D). Tubulin MTF-1 is a major transcriptional factor that protects cells from Fig. 4. XAF1–MT2A antagonism regulates cellular response to metal stress. metal stress by activating metal-responsive genes, such as MTs, (A and B) IB assays showing opposite responses of XAF1 and MT2A to cy- through the metal response element (MRE) in the promoters. – XAF1 tostatic and cytotoxic metal stresses. (C) A crucial role for XAF1 MT2A mu- Intriguingly, metal induction of mRNA was suppressed by tual antagonism in cellular response to metal stress. Cells were treated with

MTF-1 depletion, and metal-induced apoptosis was blocked by indicated doses of CdCl2 for 24 h. (D) Enhancement of metal-induced apo- either MTF-1 or XAF1 depletion (Fig. 3A and Fig. S3E). MTF-1 ptosis by WT-XAF1. (E) XAF1-mediated apoptotic switch of cellular response overexpression led to a dose-associated elevation of XAF1 to cytostatic metal stress and MT2A attenuation of apoptotic response to mRNA, and its apoptotic effect is abrogated if XAF1 induction cytotoxic metal stress.

Shin et al. PNAS | May 30, 2017 | vol. 114 | no. 22 | 5685 Downloaded by guest on September 28, 2021 A HCT116 B HCT116 on the intracellular zinc level and p53 protein, a representative si-Control si-XAF1 si-XAF1/MT2A Control ADR CPT apoptotic factor the conformation of which is regulated in a highly (0.2 M) (20 M) 1.3 0.9 0.7 Sub-G1 zinc-dependent manner (34). A spectrofluorimetric measurement 62.3 51.1 71.2 G1 5-FU TNF- IFN- Cont ET Cont ET 5-FU TNF- IFN- ET Cont 5-FU TNF- IFN- 15.6 20.1 15.1 S of Zinquin revealed that the free zinc level is increased by WT- XAF1 21.8 27.9 13.0 G2/M (%) Δ A

pcDNA XAF1 but not by ZF5-XAF1 (Fig. 6 ). Moreover, differential MT2A IB 2.5 30.5 25.6 Sub-G1 responses of XAF1 to cytostatic and cytotoxic doses were co- C. PARP 60.3 25.3 49.8 G1 Tubulin 17.3 16.4 10.1 S 19.9 27.8 14.5 G2/M (%) incident with the regulation pattern of p53 and expression of its XAF1 target genes, such as BAX, PUMA,andNOXA (Fig. S6A). The RT- MT2A PCR 1.1 0.7 1.2 Sub-G1 GAPDH 60.5 52.5 70.9 G1 XAF1 response to metals also correlated inversely with the ex- 16.4 23.1 14.2 S HCT116 22.0 29.4 13.7 G2/M (%) pression level of XIAP, an antiapoptotic factor the stability of

C ADR (0.2 M) CPT (20 M) ZF5-XAF1 WT-XAF1 which is affected by heavy metals (35, 36). Expression assays of - + + + - + + + D p53 using antibodies specific for WT (PAb1620) and mutant-type - - + - - - + - WT-XAF1 30 ** HCT116 ---+ ---+ ZF5-XAF1 25 si-Control (PAb240) p53 revealed that XAF1 expression up- and down- XAF1 si-MT2A 20 B MT2A regulates WT and mutant-type p53, respectively (Fig. 6 ). p21WAF1 15 Moreover, elevation of WT p53 conformation and its target ex- C. PARP 10 pression by cytotoxic metal stress were impeded by MT2A (Fig. 6C Tubulin 5 0 and Fig. S6B). XAF1 regulation of p53 and XIAP was not de- E HCT116 Apoptosis (sub-G1 %) -+++-+++CPT (20 M) C --+---+ -WT-XAF1 tected in MT2A-depleted cells (Fig. S6 ). IP assays also showed F ---+ ---+ ZF5-XAF1 that the 5-FU–induced conformational shift of p53 is impeded EGF TGF- 1 IL-6 IGF-1 HCT116 D - + + + + + 5-FU (25 M) when XAF1 is depleted (Fig. 6 ). The opposite effects of XAF1 si-Control si-MT2A XAF1 and MT2A on conformational shift of p53 were abolished MT2A - + + + + -+ ++ +5-FU (50 M) E D IB 0 0 10 20 50 0 0 10 20 50 EGF (ng/ml) when they were co-overexpressed (Fig. 6 and Fig. S6 ). Com- C. PARP p53+/+ p53−/− Tubulin XAF1 parative assays of HCT116 and sublines exposed to XAF1 MT2A RT- cytostatic dose of ADR revealed that the XAF1-induced apoptotic MT2A C. PARP +/+ PCR switch and its attenuation by MT2A are more drastic in p53 GAPDH Tubulin −/− versus p53 cells (Fig. 6F). Likewise, XAF1 promotion of CdCl - +/+ −/− 2 Fig. 5. XAF1–MT2A antagonism plays a key role in cellular response to induced apoptosis was more drastic in p53 versus p53 cells, nonmetal stresses. (A) A role for XAF1–MT2A interplay in cellular response to further supporting that p53 is an important target of XAF1 in genotoxic and cytokine stresses. Cells were treated with ET (50 μM), 5-FU (25 μM), evoking its proapoptotic function (Fig. S6E). TNF-α (20 ng/mL), and IFN-β (200 U/mL) for 24 h. (B and C) XAF1-mediated apoptotic switch of cellular response to ADR and CPT. (D) Abrogation of XAF1-mediated switch of cell response to CPT by MT2A depletion. (E)In- hibitory effect of growth factors on 5-FU–induced XAF1 expression. Cells AB1.6 U2OS HCT116 were pretreated with EGF (20 ng/mL), IGF1 (20 ng/mL), TGF-β1 (2 ng/mL), or HCT116 IL-6 (2 ng/mL) and exposed 5-FU for 24 h. (F) Association of antiapoptotic 1.5 WT-XAF1 ZF5-XAF1 – 1.4 function of EGF with its XAF1-repressing effect in 5-FU treated cells. p53 (PAb1620) 1.3 p53 (PAb240) 1.2 p53 (Pan-p53) (Fold change) MT2A-Targeting Function Is Closely Linked to XAF1’s Apoptosis- 1.1 XIAP Promoting Activity. – zinc Intracellular level 0 MT2A Next we assessed whether the XAF1 MT2A 0 0.5 1 2 0 0 0.5 1 2 0 WT WT-XAF1 antagonism plays a role in cellular response to nonmetal 0 0 0 0 2 0 0 0 0 2 ZF5 ZF5-XAF1 stresses. In HCT116 cells, apoptosis induction by etoposide (ET), XAF1 ( g) Tubulin α β C HCT116 D 5-fluorouracil (5-FU), TNF- , and IFN- was accompanied by GFP-MT2A HCT116 XAF1 induction and MT2A reduction (Fig. 5A and Fig. S5A). - + + + + CdCl (20 M) 2 - + - + 5-FU (25 M, 24 h) However, apoptosis induction and MT2A reduction were blocked p53 (PAb1620) - - + + si-XAF1 (40 pM) p53 (PAb240) when XAF1 expression was depleted, while apoptosis occurred WT: PAb1620 p53 (Pan-p53) IP: p53 1620 240 1620 240 1620 1620 240 240 MT: PAb240 when MT2A was codepleted. ET-induced apoptosis was strongly XIAP promoted by WT-XAF1 but not by ΔZF5-XAF1 (Fig. S5B). Upon IB: p53 (Pan-p53) MT2A (GFP) p53 (Input) exposure to adriamycin (ADR) and cisplatin (CPT), HCT116 cells XAF1 exhibited p21WAF1 but no XAF1 induction and manifested cyto- Tubulin F E HCT116 Control static response (G2 or G1 cell-cycle arrest) (Fig. 5 B and C). 40 ADR (0.2 M) ** - - + + MT2A However, this cytostatic response was switched to apoptosis when -+ -+ Flag-XAF1 30 WT-XAF1 was introduced and ΔZF5-XAF1 failed to induce this IP: p53 (PAb) 20 240 240 240 effect. Moreover, MT2A depletion caused apoptotic switch of 1620 1620 1620 240 1620 * cytostatic response, supporting that the MT2A-targeting role of IB: p53 (Pan) 10 XAF1 is crucial for its cell-fate–switching function (Fig. 5D). We p53 (Input) MT2A Aopotosis sub-G1) (% 0 next asked whether growth factors affect the XAF1–MT2A in- - - + + + + - - + + + + XAF1 XAF1 (Flag) ----++ ----++ MT2A terplay to evoke their cell protection effect. In cells exposed to WCL Tubulin p53-/- p53+/+ HCT116 5-FU, pretreatment with EGF, IGF1, TGF-β1, or IL-6 strongly inhibited XAF1 induction and MT2A reduction (Fig. 5E). Fur- Fig. 6. A XAF1-mediated free zinc increase drives a WT shift of p53 con- thermore, XAF1 inhibition and antiapoptosis effects of EGF were formation. (A) A spectrofluorimetric measurement of Zinquin showing a impaired if MT2A restoration was blocked, indicating that growth XAF1 dose-associated increment of the intracellular free zinc level. (B) Up- factors exert their prosurvival effect through the regulation of the regulation of WT p53 conformation and down-regulation of XIAP level by Δ XAF1–MT2A axis (Fig. 5F and Fig. S5C). WT-XAF1 but not by ZF5-XAF1. PAb1620 and PAb240 antibodies were used to detect WT and mutant type (under nondenaturating gel conditions) of p53 conformation, respectively. Pan-p53 antibody was used to determine XAF1 Increases the Free Intracellular Zinc Level to Promote total p53 protein level. (C) MT2A inhibition of cytotoxic metal stress-induced Conformational Change of p53. MT2A is a prototype metal- p53 conformational change and XIAP reduction. (D) IP assay showing a role binding protein that regulates intracellular zinc homeostasis, and for XAF1 in genotoxic stress-induced p53 conformational change. (E) Op- its cell-protective function is linked to zinc-scavenging activity. To posite effects of XAF1 and MT2A on p53 conformation. (F) Comparison of + + define apoptotic signaling pathways controlled by XAF1–MT2A XAF1 ability to induce apoptotic switch of ADR-treated HCT116 p53 / and antagonism, we examined the effect of XAF1–MT2A antagonism p53−/− cells.

5686 | www.pnas.org/cgi/doi/10.1073/pnas.1700861114 Shin et al. Downloaded by guest on September 28, 2021 Colon and rectum Cell lines (N=28) Discussion AB5 r2 = 0.732 P < 0.01 Upon heavy metal exposure, MT2A activated by MTF-1 se- 4 questers metals to protect cells from metal cytotoxicity (19–22). 3 Caco-2 HCT116 KM12C LoVo Colo205 Colo320 DLD-1 SNUC2A HT-29 LST174T RKO SNUC1 SW480 SW620 However, the molecular mechanism by which MT2A is controlled XAF1 2 under cytotoxic metal stress conditions remains undefined. MT2A 1 MT2A degradation takes place mainly in the lysosomes, but the

Tubulin Relative MT2A level 0 C P1 P2 P3 1 2 3 4 5 signaling pathways involved in the lysosomal trafficking of Normal Cancer Normal Cancer Normal Cancer D Relative XAF1 level MT2A have been poorly understood (33). In the present study, Colon cancers (N=35) we demonstrate first that XAF1 is a negative regulator of r2 5 = 0.594 P < 0.01 MT2A, the activation of which directs the apoptotic switch of 4 cellular response to heavy metals. XAF1 has been shown to 3 regulate protein stability through interaction with ubiquitin

MT2A XAF1 100 m 2 E3 ligases such as XIAP, cIAP1/2, ZNF313, and Siah2 (2, 12, Control CdCl 1 E 2 13, 37). Our data provide evidence that XAF1 binds to MT2A Relative MT2A level 0 1 2 3 4 5 and accelerates its lysosomal relocalization, identifying its in- HCC1937- F Relative XAF1 level volvement in both the proteasomal and lysosomal degradation shXAF1 Control CdCl 2 pathways. Intriguingly, it was also found that MT2A destabi- shXAF1 lizes XAF1 through the lysosomal pathway and that this activity HCC1937- shControl shCont is linked to its cell protection function. Therefore, our data G support the notion that XAF1–MT2A antagonism functions as )

2 CdCl HCC1937 5 HCC1937 2 H a linchpin to govern cell fate under metal stress conditions shControl shControl shXAF1 shControl shXAF1 (Fig. 8). 4 shXAF1 ------+ + + + + + CdCl2 XAF1 1 2 3 1 2 3 1 2 3 1 2 3 Tumors is a direct transcription target of p53 and IFN regulatory 3 XAF1 factor 1 (IRF-1), which is up-regulated by various apoptotic

2 ** MT2A stresses (10–12, 38). In this study, we found that XAF1 is a MT1A 1 unique transcription target of MTF-1 in signaling apoptosis and C. PARP 0 C. Casp-3 counteracts MT2A, a prosurvival target of MTF-1. Given that

Tumor volume (x 100 mm 100 Tumor volume (x 0 4 8 12 16 20 24 28 32 36 40 Tubulin MTF-1 can induce apoptosis in a highly XAF1-dependent fash- Days after inoculation ion, it is plausible that MTF-1 provokes both prosurvival and Fig. 7. Inverse correlation of XAF1 and MT2A expression in human cancers proapoptotic effects depending on its target selectivity. In this and XAF1 effect on tumor growth and response to metal stress. (A and B) context, MTF-1 might be modified differentially by cytostatic and Expression status of XAF1 and MT2A in human cancer cell lines. Relative cytotoxic stresses, allowing its selective or preferential binding to a expression levels were classified as levels 0–5. r, Pearson’s correlation co- subset of target promoters. Considering that both MTF-1 and efficient. (C) Immunohistochemical analysis of XAF1 and MT2A in human MT2A are up-regulated and that XAF1 is down-regulated in primary colon tumor and matched normal tissues. P, patient. (D) Inverse many human cancers, alteration of the MTF-1–XAF1 and/or correlation of XAF1 and MT2A immunoreactivity in colon cancer tissues. XAF1–MT2A axes might contribute to tumor resistance in heavy- Relative staining levels were classified as levels 0–5. (E–G) Mouse tumor xe- metal–induced apoptosis. nograft assays showing the XAF1 depletion effect on tumor growth and In addition to its essential role for the sequestration of heavy response to metal stress. Data represent the mean ± SD (n = 6 per group; metals away from critical macromolecules, MT2A contributes to **P < 0.01). (H) IB assay of xenograft tumor tissues showing the XAF1 de- chemotherapy resistance through the indirect action on p53 zinc- pletion effect on MT2A and cleaved PARP and caspase-3 levels. dependent activity (29, 30). MT2A is increased by HIPK2 knockdown, and this elevation correlates with p53 misfolding and cellular resistance to adriamycin-driven apoptosis (30). We XAF1 and MT2A Expression Levels Correlate Inversely in Human – have shown that XAF1 activates p53 by interfering with MDM2 Cancers. To delineate the XAF1 MT2A interpaly in human tu- interaction with p53 and also by activating HIPK2 (12). Our mors, we characterized their expression status in 28 cancer cell

present study shows that XAF1 increases the free intercellular MEDICAL SCIENCES lines of multiple tissue origins and 35 primary colon tumor tis- zinc level and activates p53 through MT2A destabilization. It is sues. A substantial fraction of cell lines and tumor tissues thus conceivable that XAF1 allows rapid amplification of the exhibited extremely low XAF1 and high MT2A expression, and a apoptotic program through Siah2-HIPK2 axis activation and strong inverse correlation was detected between their levels (Fig. 7 A–D and Fig. S7 A–C). Compared with 35 normal tissues that we examined, 10 (28.6%) and 14 (40%) tumors were shown to express abnormally low XAF1 and high MT2A (Fig. 7D and Cell cycle arrest (Life) Apoptosis (Death) Fig. S7C). Heavy metals Moreover, high MT2A was detected in 8 of 10 (80%) low- XAF1 tumors but only in 6 of 26 (23.1%) normal XAF1 tumors, Cytostatic stress Cytotoxic stress and low XAF1 was detected in 9 of 14 (64.3%) high-MT2A tu- MTF-1 mors but only in 2 of 21 (9.5%) normal MT2A tumors. Mouse Lysosome tumor xenograft assays using XAF1-depleted HCC1937 subline cells revealed that XAF1-depleted tumors show substantially MT2A XAF1 increased growth rate and delayed regression following injection of a cytotoxic dose of CdCl2 (0.05 mg/g) (Fig. 7 E–G). Compared with control tumors, XAF1-depleted tumors showed higher MTF-1 MT2A MT2A XAF1 XAF1 MTF-1 MT2A and MT1A and lower cleaved PARP and caspase-3 levels MT2A XAF1 H (Fig. 7 ). We next asked whether XAF1 induction could drive a Zn Zn Zn switch of tumor response to cytostatic metal stress using HCT116 MT2A p53 XAF1 (Tet-XAF1) xenograft tumors. Upon exposure to low dose of Zn Zn CdCl2 (0.02 mg/g), XAF1-induced tumors displayed drastic re- MT2A XIAP XAF1 gression and undetectably low MT2A whereas control tumors showed only a delayed growth and elevated MT2A expression Fig. 8. Schematic representation of the antagonistic interplay of XAF1 and (Fig. S7 D–F). MT2A under metal stress conditions and its role in cell-fate decisions.

Shin et al. PNAS | May 30, 2017 | vol. 114 | no. 22 | 5687 Downloaded by guest on September 28, 2021 MT2A inactivation. Consistently, we observed that the cell-fate Collectively, this study demonstrates that XAF1–MT2A mu- decision function of the XAF1–MT2A axis is more drastic in tual antagonism represents one crucial molecular switch in cell- +/+ −/− p53 versus isogenic p53 cells. fate decisions under stressful conditions, adding a layer of XAF1 is originally identified to interact with and sequester complexity to the mechanisms by which stress response is regu- XIAP to the nucleus (2, 3). However, XAF1’s proapoptotic lated. Our data suggest that disruption of XAF1–MT2A in- function is not solely dependent on the XIAP-interfering activity terplay may contribute to the development and progression of (6, 9). Our study shows that XAF1 induction by heavy metals various diseases, including malignant tumors. leads to a drastic reduction of XIAP in a highly MT2A-dependent manner. Studies have shown that intracellular copper accumula- Materials and Methods tion causes XIAP reduction through the copper-induced confor- Details regarding human tissues, cancer cell lines, cellular assays of cell cycle mational change and that cadmium down-regulates XIAP via a and apoptosis, expression constructs, ChIP, immunofluorescence, immuno- proteasome-mediated mechanism, and this reduction coincides with blotting, GST pull-down, measurement of intracellular zinc concentration, an increased cellular sensitivity to TNF-α–induced apoptosis (35, and semiquantitative RT-PCR analysis are available in SI Materials and 36). Given that MT2A presents a high affinity for copper, our study Methods. All animal studies were performed with the approval of Korea raises the possibility that XAF1-mediated MT2A destabilization University Institutional Animal Care and Use Committee and Korea Animal increases the free intracellular copper level and thereby leads to Protection Law. conformational change and degradation of XIAP, lowering the ACKNOWLEDGMENTS. We thank Dr. Bert Vogelstein (Johns Hopkins Univer- + + − − apoptotic threshold and sensitizing the cell to apoptosis (39). Our sity) for HCT116 p53 / and p53 / cells. This work was supported in part by study thus identifies an alternative mechanism of XAF1 inhibition National Research Foundation of Korea Grants NRF-2015R1A2A1A01005389 of XIAP by attenuating MT2A stabilization of XIAP. (to S.-G.C.) and NRF-2015R1D1A401016836 (to M.-G.L.).

1. Roos WP, Thomas AD, Kaina B (2016) DNA damage and the balance between survival 21. McGee HM, Woods GM, Bennett B, Chung RS (2010) The two faces of metallothionein and death in cancer biology. Nat Rev Cancer 16:20–33. in carcinogenesis: Photoprotection against UVR-induced cancer and promotion of 2. Fong WG, et al. (2000) Expression and genetic analysis of XIAP-associated factor 1 tumour survival. Photochem Photobiol Sci 9:586–596. (XAF1) in cancer cell lines. Genomics 70:113–122. 22. Theocharis SE, Margeli AP, Koutselinis A (2003) Metallothionein: A multifunctional 3. Liston P, et al. (2001) Identification of XAF1 as an antagonist of XIAP anti-Caspase protein from toxicity to cancer. Int J Biol Markers 18:162–169. – activity. Nat Cell Biol 3:128 133. 23. Shi Y, et al. (2010) The metal-responsive transcription factor-1 protein is elevated in 4. Byun DS, et al. (2003) Hypermethylation of XIAP-associated factor 1, a putative tumor human tumors. Cancer Biol Ther 9:469–476. suppressor gene from the 17p13.2 locus, in human gastric adenocarcinomas. Cancer 24. Theocharis SE, Margeli AP, Klijanienko JT, Kouraklis GP (2004) Metallothionein ex- – Res 63:7068 7075. pression in human neoplasia. Histopathology 45:103–118. 5. Lee MG, et al. (2006) Promoter CpG hypermethylation and downregulation of 25. Cherian MG, Jayasurya A, Bay BH (2003) Metallothioneins in human tumors and XAF1 expression in human urogenital malignancies: Implication for attenuated potential roles in carcinogenesis. Mutat Res 533:201–209. p53 response to apoptotic stresses. Oncogene 25:5807–5822. 26. Ma H, et al. (2015) HMBOX1 interacts with MT2A to regulate autophagy and apo- 6. Chung SK, et al. (2007) Frequent alteration of XAF1 in human colorectal cancers: ptosis in vascular endothelial cells. Sci Rep 5:15121. Implication for tumor cell resistance to apoptotic stresses. Gastroenterology 132: 27. Krzeslak A, et al. (2013) Effect of metallothionein 2A gene polymorphism on allele- 2459–2477. specific gene expression and metal content in prostate cancer. Toxicol Appl 7. Lunardi A, et al. (2013) A co-clinical approach identifies mechanisms and potential – therapies for androgen deprivation resistance in prostate cancer. Nat Genet 45: Pharmacol 268:278 285. 747–755. 28. Krzeslak A, et al. (2014) Metallothionein 2A genetic polymorphisms and risk of ductal – 8. Ng KCP, Campos EI, Martinka M, Li G (2004) XAF1 expression is significantly reduced breast cancer. Clin Exp Med 14:107 113. ’ in human melanoma. J Invest Dermatol 123:1127–1134. 29. Habel N, et al. (2013) Zinc chelation: A metallothionein 2A s mechanism of action 9. Xia Y, Novak R, Lewis J, Duckett CS, Phillips AC (2006) Xaf1 can cooperate with involved in osteosarcoma cell death and chemotherapy resistance. Cell Death Dis TNFalpha in the induction of apoptosis, independently of interaction with XIAP. Mol 4:e874. Cell Biochem 286:67–76. 30. Puca R, et al. (2009) Restoring wtp53 activity in HIPK2 depleted MCF7 cells by mod- 10. Leaman DW, et al. (2002) Identification of X-linked inhibitor of apoptosis-associated ulating metallothionein and zinc. Exp Cell Res 315:67–75. factor-1 as an interferon-stimulated gene that augments TRAIL Apo2L-induced apo- 31. Garrett SH, et al. (2000) Metallothionein isoform 1 and 2 gene expression in the ptosis. J Biol Chem 277:28504–28511. human prostate: Downregulation of MT-1X in advanced prostate cancer. Prostate 43: 11. Micali OC, et al. (2007) Silencing of the XAF1 gene by promoter hypermethylation in 125–135. cancer cells and reactivation to TRAIL-sensitization by IFN-beta. BMC Cancer 7:52–59. 32. Jin R, et al. (2002) Metallothionein 2A expression is associated with cell proliferation 12. Lee MG, et al. (2014) XAF1 directs apoptotic switch of p53 signaling through acti- in breast cancer. Carcinogenesis 23:81–86. vation of HIPK2 and ZNF313. Proc Natl Acad Sci USA 111:15532–15537. 33. Klaassen CD, Choudhuri S, McKim JM, Jr, Lehman-McKeeman LD, Kershaw WC (1994) 13. Han J, et al. (2013) ZNF313 is a novel cell cycle activator with an E3 ligase activity In vitro and in vivo studies on the degradation of metallothionein. Environ Health WAF1 inhibiting cellular senescence by destabilizing p21( .). Cell Death Differ 20: Perspect 102:141–146. – 1055 1067. 34. Kruse JP, Gu W (2009) Modes of p53 regulation. Cell 137:609–622. 14. Zhu LM, et al. (2014) Tumor suppressor XAF1 induces apoptosis, inhibits angiogenesis 35. Mufti AR, et al. (2006) XIAP is a copper binding protein deregulated in Wilson’s dis- – and inhibits tumor growth in hepatocellular carcinoma. Oncotarget 5:5403 5415. ease and other copper toxicosis disorders. Mol Cell 21:775–785. 15. Wang J, et al. (2009) Identification of XAF1 as a novel cell cycle regulator through 36. Golovine K, et al. (2010) Cadmium down-regulates expression of XIAP at the post- modulating G(2)/M checkpoint and interaction with checkpoint kinase 1 in gastro- transcriptional level in prostate cancer cells through an NF-kappaB-independent, intestinal cancer. Carcinogenesis 30:1507–1516. proteasome-mediated mechanism. Mol Cancer 9:183. 16. Bishak YK, Payahoo L, Osatdrahimi A, Nourazarian A (2015) Mechanisms of cadmium 37. Arora V, et al. (2007) Degradation of survivin by the X-linked inhibitor of apoptosis carcinogenicity in the gastrointestinal tract. Asian Pac J Cancer Prev 16:9–21. (XIAP)-XAF1 complex. J Biol Chem 282:26202–26209. 17. Kimura T, Kambe T (2016) The functions of metallothionein and ZIP and ZnT trans- 38. Wang J, et al. (2009) c-Jun N-terminal kinase (JNK1) upregulates XIAP-associated porters: An overview and perspective. Int J Mol Sci 17:336. 18. Heger Z, et al. (2016) Metallothionein as a scavenger of free radicals: New car- factor 1 (XAF1) through interferon regulatory factor 1 (IRF-1) in gastrointestinal cancer. – dioprotective therapeutic agent or initiator of tumor chemoresistance? Curr Drug Carcinogenesis 30:222 229. Targets 17:1438–1451. 39. Nakamura S, et al. (2004) Implication of the differential roles of metallothionein 19. Suzuki K, Koizumi S (2000) Individual metal responsive elements of the human 1 and 2 isoforms in the liver of rats as determined by polyacrylamide-coated capillary metallothionein-IIA gene independently mediate responses to various heavy metal zone electrophoresis. Biochem Biophys Res Commun 320:1193–1198. signals. Ind Health 38:87–90. 40. Lee MG, et al. (2016) RASSF1A directly antagonizes RhoA activity through the as- 20. Takahashi S (2015) Positive and negative regulators of the metallothionein gene sembly of a Smurf1-mediated destruction complex to suppress tumorigenesis. (review). Mol Med Rep 12:795–799. Cancer Res 76:1847–1859.

5688 | www.pnas.org/cgi/doi/10.1073/pnas.1700861114 Shin et al. Downloaded by guest on September 28, 2021