Regulation of hypoxia responses by flavin adenine dinucleotide-dependent modulation of HIF-1a protein stability

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1 Article Regulation of hypoxia responses by flavin adenine dinucleotide-dependent modulation of HIF-1a protein stability Suk-Jin Yang 1,2, Young Soo Park 1,3, Jung Hee Cho 4, Byul Moon 1,3, Hyun-Jung Ahn 4, Ju Yeon Lee 5, Zhi Xie 6, Yuli Wang 6, David Pocalyko 6, Dong Chul Lee 1, Hyun Ahm Sohn 1, Minho Kang 4, Jin Young Kim 5, Eunhee Kim 2, Kyung Chan Park 3,4,*, Jung-Ae Kim 3,4,** & Young Il Yeom 1,3,*** Abstract Oxygen deprivation induces a range of cellular adaptive responses that enable to drive cancer progression. Here, we report that lysine-specific demethylase 1 (LSD1) upregulates hypoxia responses by demethylating RACK1 protein, a component of hypoxia-inducible factor (HIF) ubiquitination machinery, and consequently suppressing the oxygen-independent degradation of HIF-1a. This ability of LSD1 is attenuated during prolonged hypoxia, with a decrease in the cellular level of flavin adenine dinucleotide (FAD), a metabolic cofactor of LSD1, causing HIF-1a downregulation in later stages of hypoxia. Exogenously provided FAD restores HIF-1a stability, indicating a rate-limiting role for FAD in LSD1-mediated HIF-1a regulation. Transcriptomic analyses of patient tissues show that the HIF-1 signature is highly correlated with the expression of LSD1 target genes as well as the enzymes of FAD biosynthetic pathway in triple-negative breast cancers, reflecting the significance of FAD-dependent LSD1 activity in cancer progression. Together, our findings provide a new insight into HIF-mediated hypoxia response regulation by coupling the FAD dependence of LSD1 activity to the regulation of HIF-1a stability. Keywords cancer; FAD biosynthesis; HIF-1; hypoxia; LSD1 Subject Categories Cancer; Metabolism; Post-translational Modifications, Proteolysis & Proteomics DOI /embj Received 23 March 2016 Revised 24 January 2017 Accepted 26 January 2017 Introduction An accumulating body of literature reports that a number of chromatin regulators, including various histone modification enzymes, are involved in different stages of tumorigenesis. Both genetic alterations and aberrant expression of chromatin regulators are known to alter the chromatin landscape and thereby affect cancer cell proliferation and/or differentiation (Dawson & Kouzarides, 2012). Recent studies show that the availability of metabolic cofactors necessary for the enzymatic activity of chromatin regulators also affects cancer progression (Gut & Verdin, 2013). Furthermore, a growing list of non-histone substrates for histone-modifying enzymes expands the functional scope of chromatin regulators beyond changing chromatin structure and toward regulating diverse signaling pathways involved in different cellular functions (Hamamoto et al, 2015). Given these findings, targeting of chromatin regulators has emerged as a promising anti-cancer therapeutic strategy. Lysine-specific demethylase 1 (LSD1) is a histone-modifying enzyme that removes mono- and di-methyl groups from lysine 4 or lysine 9 on H3 via a flavin adenine dinucleotide (FAD)-dependent oxidative reaction (Lan et al, 2008). Mono- and di-methylation of H3K4 (H3K4me1/2) is strongly associated with transcriptional activation, whereas di-methylation of H3K9 (H3K9me2) is involved in a repressive mode of transcription (Zhou et al, 2011). Due to the dual specificity toward H3K4me1/2 and H3K9me1/2, the effect of LSD1 on target gene expression appears to be context-dependent. Indeed, LSD1 is known to physically associate with multiple transcriptional regulators including NuRD, Co-REST, AR, and AML (Nakamura et al, 2002; Metzger et al, 2005; Shi et al, 2005; Wang et al, 2009b). In conjunction with specific binding partners, LSD1 regulates the transcription of distinct sets of target genes. Furthermore, LSD1 is 1 Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, South Korea 2 Department of Bioscience and Biotechnology, Chungnam National University, Daejeon, South Korea 3 Department of Functional Genomics, University of Science and Technology, Daejeon, South Korea 4 Personalized Genomic Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, South Korea 5 Biomedical Omics Group, Korea Basic Science Institute, Cheongju, South Korea 6 Pfizer Global Research and Development, San Diego, CA, USA *Corresponding author. Tel: ; kpark@kribb.re.kr **Corresponding author. Tel: ; jungaekim@kribb.re.kr ***Corresponding author. Tel: ; yeomyi@kribb.re.kr ª 2017 The Authors The EMBO Journal 1

2 The EMBO Journal Hypoxia responses regulated by FAD-dependent LSD1 Suk-Jin Yang et al known to regulate the methylation dynamics of non-histone proteins. The di-methyl group at lysine 370 on p53, which facilitates the binding of 53BP1, is erased by LSD1, causing the downregulation of p53-responsive genes (Huang et al, 2007). In addition, the stability of DNMT, E2F, and MYPT1 proteins is regulated by the non-histone demethylase activity of LSD1 (Wang et al, 2009a; Kontaki & Talianidis, 2010; Cho et al, 2011). These findings highlight that LSD1 has diverse physiological roles, acting not only as a chromatin modifier but also as a signaling regulator for nonchromatin events. Aberrant expression of LSD1 has been shown in many cancer types including blood, neuronal, prostate, lung, colorectal, bladder, pancreatic, and breast cancers (Amente et al, 2013), implicating LSD1 as a cancer biomarker. In acute myeloid leukemia (AML), a type of blood cancer, LSD1 bound to MLL supercomplex regulates the expression of the MLL-mediated oncogenic program by modulating H3 methylation (Harris et al, 2012). In line with these observations, either genetic or pharmacological inhibition of LSD1 suppresses oncogenic stem cell characteristics (Harris et al, 2012; Schenk et al, 2012), highlighting the therapeutic potential of targeting LSD1 for AML treatment. However, despite the strong correlation of LSD1 expression with cancerous incidence and poor prognosis, the mechanism of LSD1-mediated solid cancer progression remains elusive. The rapid growth of solid tumors frequently generates oxygendeprived microenvironments due to inefficient oxygen delivery from distal blood vessels (Bristow & Hill, 2008). Chromatin regulators modulating histone acetylation or methylation appear to affect hypoxic responses by serving as transcriptional cofactors of hypoxia-inducible factors (HIFs) (Zhong et al, 2010; Mimura et al, 2012). Moreover, histone lysine deacetylases such as SIRT1 and HDAC4 are suggested to modulate the acetylation dynamics of HIF-1a protein (Lim et al, 2010a; Geng et al, 2011). Conversely, hypoxia can modulate the enzymatic activity of chromatin regulators per se by changing the amount of their substrates and metabolic cofactors. For instance, the lysine deacetylase activities of SIRT1 and SIRT6 are downregulated by a reduced NAD + /NADH ratio in hypoxic cells (Lim et al, 2010a; Zhong et al, 2010), and the enzymatic activity of JmjC domain-containing histone lysine demethylases (KDMs), such as KDM3A, decreases in hypoxia due to the low concentration of oxygen (Xia et al, 2009). However, unlike most KDMs, LSD1 (also known as KDM1A), which lacks the JmjC domain, does not require oxygen as its co-substrate (Lan et al, 2008), and the molecular mechanism of its involvement in the hypoxic response remains to be clearly defined. In this study, given that LSD1 is strongly associated with solid cancer progression, which is frequently accompanied by hypoxia, we investigated the molecular functions of LSD1 in hypoxic cancer cells. First, we demonstrate that HIF-1a is stabilized when LSD1 demethylates RACK1 protein, a key component of oxygen-independent degradation of HIF-1a subunits. We also provide evidence that a change in cellular FAD level is a critical factor determining HIF-1a stability during hypoxia, as it modulates LSD1-dependent RACK1 demethylation. Furthermore, we demonstrate the prognostic significance of the association among FAD biosynthetic enzyme expression, LSD1-target gene expression and HIF-1 signature gene expression in a cancer cohort. Taken together, these findings lead us to propose the biological significance of FAD metabolism and LSD1 activity in HIF-mediated hypoxic responses and their contribution to cancer progression. Results The lysine demethylase activity of LSD1 is required for the accumulation of HIF-1a protein in hypoxia To investigate the role of LSD1 in the cell proliferation of solid cancers, we estimated the sensitivity of 50 different cancer cell lines to sirna-mediated depletion of LSD1 and stratified them into three groups based on their dependence on LSD1 for cell proliferation (Table EV1 and Appendix Fig S1A and B). Cells displaying more than 65% of proliferation defects by LSD1 depletion were classified as Class I, while those exhibiting < 35% of defects were grouped as Class III. The remaining cells were categorized as Class II. We then determined the gene expression profile of representative cell lines from each group by microarray analyses and identified molecular features closely correlated with the LSD1 dependence of cell proliferation. Gene set enrichment analyses (GSEA) showed that among others, glycolysis was particularly highly enriched in Class I compared with Class III (Figs 1A and EV1A). This result was reproducible regardless of the tissue origin of different cancer cells. In fact, when human hepatocellular carcinoma (HCC) cells analyzed for the GSEA were examined, Class I cells displayed significantly higher average glycolysis activities than Class III cells as experimentally confirmed by measuring the levels of glucose uptake and lactate production (Fig 1B). These results indicate that cells with higher LSD1 dependence tend to exhibit higher glycolytic activity, thus leading us to hypothesize that LSD1 may have an important role in the glycolysis-dependent proliferation of cancer cells. To test this hypothesis, we first examined the effect of LSD1 knockdown (KD) on cell proliferation under hypoxic conditions in which cellular glycolytic activities are upregulated by way of metabolic adaptation. We found that depletion of LSD1 significantly impaired cell proliferation, especially under hypoxia, with a tendency of Class I cells (Huh-1, NCI-H596) more significantly affected than Class III cells (PLC/PRF/5, Colo-205; Fig 1C). Next, we determined the involvement of LSD1 in the elevation of glycolysis under hypoxia in these cell lines. Consistent with its effect on cell growth, LSD1 KD resulted in a significant reduction in both glucose uptake and lactate production in hypoxia (Fig EV1B and C). These results suggest that LSD1 positively regulates glycolytic activity in hypoxia. Therefore, we examined the direct role of LSD1 in the regulation of hypoxic glycolysis. HIF-1, known as the master regulator of hypoxia responses (Keith et al, 2012), plays a pivotal role in the upregulation of glycolysis. We first measured the effect of LSD1 on the activity of the 5 HRE (hypoxia-responsive element) reporter carrying five tandem repeats of the HIF binding site in HEK293T cells. LSD1 KD significantly decreased HRE reporter activity both in normoxia and in hypoxia (Fig EV1D). Consistently, sirna-mediated depletion of LSD1 resulted in a dramatic reduction of HIF-1a protein accumulation under hypoxia in various cell types (Fig 1D and Appendix Fig S1C and D). Similar results were observed when cells were treated with the hypoxia-mimetic CoCl 2 (Appendix Fig S1E). By contrast, in cells depleted of LSD1 by shrna targeting the 3 0 UTR of LSD1, the hypoxic induction of HIF-1a protein was rescued 2 The EMBO Journal ª 2017 The Authors

3 Suk-Jin Yang et al Hypoxia responses regulated by FAD-dependent LSD1 The EMBO Journal A B C D E F Figure 1. ª 2017 The Authors The EMBO Journal 3

4 The EMBO Journal Hypoxia responses regulated by FAD-dependent LSD1 Suk-Jin Yang et al Figure 1. The lysine demethylase activity of LSD1 is required for hypoxic accumulation of HIF-1a protein. A Gene set enrichment analysis (GSEA) showing the enrichment of glycolysis pathway in the Class I as compared with Class III. Ten cell lines from Class I (HLF, JHH-6, Huh-1, Malme-3M, JHH-4, NCI-H1299, NCI-H596, MIA-PaCa-2, SK-HEP-1, and SNU-475) and nine cell lines from Class III (SNU-739, SNU-423, SNU-886, Calu-1, BxPC-3, Capan-2, PLC/PRF/5, AsPC-1, and Hep3B) were analyzed (left; various tissues). HCC cells from Class I (HLF, JHH-6, Huh-1, JHH-4, SK-HEP-1, and SNU-475) and Class III (SNU-739, SNU-423, SNU-886, PLC/PRF/5, and Hep3B) were also analyzed (right; liver only). NES: normalized enrichment score, FDR: false discovery rate. B Assessment of glycolytic activities in five representative cell lines from either HCC Class I (red; HLF, JHH-6, Huh-1, JHH-4, SK-HEP-1, and SNU-475) or Class III (blue; SNU-739, SNU-423, SNU-886, PLC/PRF/5, and Hep3B). Glucose uptake (left) and lactate production (right) were measured by detecting fluorescence at Ex/Em = 530/ 590 and colorimetric absorbance at OD 565 nm, respectively. Values are means SD of biological triplicate samples. Student s t-test was performed to assess statistical significance indicated as P-values. C Effects of LSD1 silencing on the proliferation of Class I (Huh-1, NCI-H596) and Class III (PLC/PRF/5, Colo-205) cells. Cells transfected with indicated sirna were incubated for 24 h in normoxia, cultured further for 96 h in normoxia (21% O 2 ) or under hypoxic conditions (3% O 2 ), and then, relative cell viabilities were measured by MTT assays. Values are means SD of biological triplicate experiments. P-values were determined by Student s t-test. D HIF-1a expression in LSD1-depleted cells under hypoxic conditions (1% O 2, 8 h). The expression levels of indicated proteins and mrnas were detected by immunoblotting (IB) and RT PCR, respectively. E Effect of LSD1 enzymatic activity on the hypoxic (1% O 2, 4 h) expression of HIF-1a protein. HEK293T cells ectopically expressing either wild-type (WT) or catalytically dead mutant (K661A) of LSD1 were determined for HIF-1a expression by IB and RT PCR. F Effect of the pharmacological inhibition of LSD1 enzymatic activity on the HIF-1a protein level in NCI-H596 cells. Cells were pre-treated with 100 lm clorgyline, 4 mm pargyline, or 2 mm tranylcypromine for 24 h, subsequently cultured under hypoxic conditions (1% O 2, 8 h), and then assessed for the HIF-1a protein level by IB. Source data are available online for this figure. by the ectopic expression of LSD1 (Appendix Fig S1F), indicating the direct involvement of LSD1 in the regulation of HIF-1a protein accumulation. Supporting these observations, LSD1 depletion caused a significant reduction in HIF-1 target gene expression including those involved in glycolysis (Fig EV1E and Appendix Fig S1G). Conversely, overexpression of LSD1 increased ectopic HIF-1a protein expression even in normoxia, without affecting its mrna level (Fig EV1F). In accordance, ectopic expression of wild-type LSD1 along with HIF-1a enhanced HIF-mediated activation of the HRE reporter in normoxia (Fig EV1H). These results raise the possibility that LSD1 has a direct function in the accumulation of HIF-1a irrespective of oxygen. By contrast, a catalytically dead mutant of LSD1, LSD1 (K661A), failed to increase HIF-1a protein level in hypoxic cells (Figs 1E and EV1G) or facilitate HIF transcriptional activity (Fig EV1H). LSD1 inhibitors also blocked the hypoxic accumulation of HIF-1a (Fig 1F) and the expression of HIF-1 target genes under hypoxia (Appendix Fig S1H) at concentrations not affecting cell viability (Appendix Fig S1I and J). Together, these results demonstrate the essential role of LSD1 and its demethylase activity in the accumulation of HIF-1a protein and the upregulation of glycolysis in hypoxia. LSD1 regulates HIF-1a protein stability in a RACK1- dependent manner The fact that LSD1 depletion blocks HIF-1a protein accumulation under hypoxia without affecting its mrna expression (Figs 1 and EV1, and Appendix Fig S1) indicates that LSD1-mediated HIF-1a regulation is achieved at the post-transcriptional level. We therefore examined the molecular mechanism of LSD1 action by investigating its effects on the stability of HIF-1a protein. Inhibition of proteasomal degradation by MG132 partially restored the accumulation of HIF-1a protein in LSD1-depleted NCI-H596 cells (Fig 2A). This result suggests that LSD1 is involved in suppressing the proteasomal degradation of HIF-1a. We then examined whether LSD1 interferes with the oxygen-dependent HIF-1a degradation mediated by von Hippel Lindau (VHL) protein. Ablation of VHL did not restore the HIF-1a protein level in LSD1-depleted NCI-H596 cells under hypoxia (Fig 2B). In addition, decreased hypoxic expression of HIF-1a protein was still observed in RCC4 cells lacking functional VHL, in which LSD1 function was genetically or pharmacologically blocked (Fig 2C and D). Note that the LSD1 inhibitors had little cytotoxic effects on RCC4 cells at the test concentrations (Fig EV2A and B). These results suggest that LSD1-dependent HIF-1a stability is not mediated by the VHL-dependent pathway. Moreover, ectopic expression of both wild-type HIF-1a and a mutant that was rendered resistant to the oxygen-induced proteasomal degradation by introducing proline-to-alanine substitutions at proline residues 402 and 564 (P/A) was also suppressed by LSD1 depletion (Fig 2E). These results, along with those for LSD1 overexpression in normoxia (Fig EV1F and H), indicate that LSD1 stabilizes HIF-1a protein via an oxygen-independent pathway that does not require VHL. Therefore, we examined whether LSD1 is involved in either CHIP-Hsp70 or RACK1-Hsp90 pathways, both of which are the oxygen-independent mechanisms of HIF-1a degradation (Liu et al, 2007; Luo et al, 2010). We found that double KD of LSD1 along with RACK1 but not with CHIP restored the hypoxia-induced HIF-1a protein level (Fig 2F). RACK1 KD also restored HIF-1a protein level in the cells treated with the LSD1 inhibitor, clorgyline under hypoxia (Fig 2G). These results demonstrate that LSD1 negatively regulates the RACK1-mediated HIF-1a degradation pathway. Interestingly, accumulation of HIF-2a protein, another substrate of the RACK1- mediated degradation machinery (Liu et al, 2007), was also compromised upon LSD1 depletion in 786-O cells, which are genetically null for VHL and HIF-1a (Fig EV2C), whereas LSD1 overexpression increased endogenous HIF-2a protein in normoxic HEK293T cells (Fig EV2D). Collectively, these results indicate that LSD1 promotes the accumulation of HIF-1a and possibly HIF-2a proteins by suppressing their oxygen-independent degradation via the RACK1-Hsp90 pathway. LSD1 specifically regulates the methylation of lysine 271 residue on RACK1 We next investigated the molecular mechanism by which LSD1 suppresses the RACK1-Hsp90 pathway in more detail. 4 The EMBO Journal ª 2017 The Authors

5 Suk-Jin Yang et al Hypoxia responses regulated by FAD-dependent LSD1 The EMBO Journal A B C D E F G Figure 2. ª 2017 The Authors The EMBO Journal 5

6 The EMBO Journal Hypoxia responses regulated by FAD-dependent LSD1 Suk-Jin Yang et al Figure 2. LSD1 inhibits the RACK1-dependent degradation of HIF-1a protein. A Effect of blocking proteasomal activity on the HIF-1a protein levels in LSD1-depleted cells. NCI-H596 cells were cultured either in normoxia (21% O 2 ) or under hypoxic conditions (1% O 2 ) with or without MG132 (10 lm) for 4 h and then subsequently subjected to measuring protein levels by IB with the indicated antibodies. B Effect of silencing VHL on the HIF-1a protein level in LSD1-depleted NCI-H596 cells under hypoxic conditions (1% O 2, 8 h). The expression levels of indicated proteins and mrnas were detected by IB and RT PCR, respectively. C Regulation of HIF-1a protein expression by LSD1 in RCC4 (VHL / ) cells under either normoxic or hypoxic (1% O 2, 8 h) conditions. Protein levels were determined by IB with the indicated antibodies. D Effect of LSD1 inhibitors (C: 100 lm clorgyline, P: 4 mm pargyline, or T: 1 mm tranylcypromine, 16 h) on the accumulation of HIF-1a in normoxic RCC4 cells. Protein levels were determined by IB against the indicated antibodies. E Examination of the oxygen independence of the HIF-1a protein regulation by LSD1. The levels of either wild-type (WT) or mutant (P/A: P402A, P564A) form of Myctagged HIF-1a ectopically expressed in LSD1-depleted HEK293T cells in normoxia were measured by IB. F Examination of the role of CHIP-Hsp70 and RACK1-Hsp90 pathways in the LSD1-mediated HIF-1a regulation. Hypoxic (1% O 2, 8 h) accumulation of HIF-1a protein in NCI-H596 cells co-transfected with silsd1 along with either sirack1 or sichip was monitored by IB analysis. mrna levels were determined by RT PCR. G Examination of the role of CHIP-Hsp70 and RACK1-Hsp90 pathways in HIF-1a regulation using an LSD1 inhibitor. Hypoxic (1% O 2, 8 h) accumulation of HIF-1a protein in either RACK1- or CHIP-depleted NCI-H596 cells pre-treated with or without clorgyline (100 lm, 24 h) was monitored by IB analysis. mrna levels were determined by RT PCR. Source data are available online for this figure. Co-immunoprecipitation assays using HEK293T cells ectopically expressing RACK1 and LSD1 showed that LSD1 physically associates with RACK1 (Fig 3A). Consistently, endogenous RACK1 but not CHIP was detected in the immunoprecipitation complex for LSD1 that was ectopically expressed in HEK293T cells (Fig EV3A and B). We also observed the endogenous binding of LSD1 to RACK1 by detecting LSD1 in the endogenous RACK1 complex immunoprecipitated from either HEK293T or NCI-H596 cells (Fig 3B). In vitro binding between recombinant GST-LSD1 and His-RACK1 proteins indicated that the two proteins directly interact with each other without an involvement of additional components (Fig EV3C). Despite the well-defined role of LSD1 as a transcriptional regulator, transcription of RACK1 and Hsp90 was not affected by LSD1 depletion (Fig EV3D). These results raise the possibility that LSD1 posttranscriptionally regulates RACK1 protein via its lysine demethylase activity. Examination by immunoprecipitating Flag-tagged RACK1 from HEK293T cells, followed by immunoblotting with anti-panmethyl-lysine antibody, indicated that RACK1 was indeed methylated in vivo and that its methylation level was downregulated by LSD1 overexpression (Fig 3C). By contrast, LSD1 KD increased methylation on RACK1 (Fig 3D). As the methylation of RACK1 protein has not been reported previously, we carried out tandem mass spectrometry analyses on RACK1 immunoprecipitated from HEK293T cells and found that RACK1 is di-methylated at lysine 271 residue (K271; Fig 3E). Supporting this finding, substitution of the K271 residue to alanine, but not the K172 residue, another candidate methylatable residue found by mass spectrometry analysis, prominently decreased the methylation signal of ectopically expressed RACK1 (Fig 3F). A polyclonal antibody directed against K271 dimethylated peptide (Figs 3G and EV3E) detected the di-methylation of K271 on RACK1 (RACK1K271me2) ectopically expressed in HEK293 cells (Fig 3H). The RACK1K271me2 signal was dramatically decreased by LSD1 overexpression in HEK293T cells, whereas it was increased, either coming from ectopically expressed or endogenous RACK1, by genetic depletion or pharmacological inhibition of LSD1 (Figs 3I and J, and EV3F and G). These results strongly suggest that methylation at K271 on RACK1 might be directly regulated by LSD1. To examine whether LSD1 indeed directly demethylates RACK1K271me2, we performed an in vitro LSD1 assay employing recombinant LSD1 protein and synthetic peptides encompassing the amino acid region on RACK1. LSD1 demethylated the RACK1 peptide carrying K271me2 (RACK1K271me2) as well as the positive control peptide carrying di-methylation of the K4 residue of histone H3 (H3K4me2; Fig 3K). By contrast, no LSD1 activity was observed toward the peptides mutrack1k271me2 (carrying alanine substitution mutations adjacent to K271 residue) and RACK1K172me2 (encompassing the amino acid region on RACK1 with di-methylation at K172 residue), indicating that the lysine demethylase activity of LSD1 is specific to RACK1K271me2 in the given sequence context. This result reinforces that RACK1K271me2 is a bona fide non-histone substrate of the lysine demethylase activity of LSD1. LSD1-mediated demethylation of RACK1K271me2 inhibits physical interaction between RACK1 and HIF-1a, suppressing HIF-1a protein turnover Next, we examined the biological consequence of the LSD1- mediated demethylation of RAKC1K271me2, that is, its effect on HIF-1a protein stability. Because RACK1 brings the Elongin C- containing E3 ubiquitin ligase complex to HIF-1a, leading to its proteasomal degradation, overexpression of RACK1 is known to reduce cellular HIF-1a protein level in hypoxia (Liu et al, 2007). However, we found that unlike the ectopic expression of wild-type RACK1, mutant RACK1(K271A) failed to decrease HIF-1a protein level in hypoxic cells (Fig 4A). This result suggests that K271 is a critical residue for RACK1-mediated degradation of HIF-1a. An in vivo ubiquitination assay employing an HA-tagged ubiquitin expression construct showed that whereas the ectopic expression of wild-type RACK1 enhanced HIF-1a ubiquitination, RACK1(K271A) mutant failed to do so (Fig 4B), suggesting that K271 methylation of RACK1 plays a critical role in the ubiquitination-mediated degradation of HIF-1a. As K271 is adjacent to the WD6 domain on RACK1, which is necessary for its association with HIF-1a, we determined whether LSD1 controls the physical interaction between RACK1 and HIF-1a by regulating the methylation of RACK1K271me2. In coimmunoprecipitation assays, we observed that depletion of LSD1 dramatically enhanced the physical interaction between wild-type RACK1 and HIF-1a, whereas the binding of mutant RACK1(K271A) to HIF-1a was negligible compared to that of wild-type RACK1 and 6 The EMBO Journal ª 2017 The Authors

7 Suk-Jin Yang et al Hypoxia responses regulated by FAD-dependent LSD1 The EMBO Journal A B C D E F G H I J K Figure 3. ª 2017 The Authors The EMBO Journal 7

8 The EMBO Journal Hypoxia responses regulated by FAD-dependent LSD1 Suk-Jin Yang et al Figure 3. LSD1 demethylates RACK1 protein at K271 residue. A Identification of LSD1-RACK1 interaction by reciprocal co-immunoprecipitations of ectopically expressed proteins from HEK293T cells. B Identification of physical interaction between endogenous RACK1 and LSD1 proteins. The interaction was detected by immunoprecipitating endogenous RACK1 from either HEK293T or NCI-H596 cells, followed by IB with an anti-lsd1 antibody. C Identification of the LSD1-mediated regulation of RACK1 methylation. RACK1 protein methylation was monitored by immunoprecipitating ectopically expressed RACK1 from HEK293T cells with or without LSD1 overexpression, followed by IB analysis with the indicated antibodies including anti-pan-methyl-lysine antibody (Pan-Me-K). D RACK1 protein methylation in LSD1-depleted HEK293T cells was monitored as described in (C). E Mass spectrometric analysis of RACK1 immunoprecipitated from HEK293T cells indicates RACK1 methylation at K271 residue. F Experimental confirmation of the methyl-lysine residue of RACK1 that is targeted by LSD1. Wild-type (WT) and mutant forms (K172A ork271a) of RACK1 proteins were ectopically expressed in HEK293T cells, immunoprecipitated using an anti-flag antibody and then determined for their methylation content as described in (C). G Dot-blot analysis of RACK1K271 peptides carrying mono- (me1), di- (me2), or tri-(me3) methylation at the K271 residue as well as the non-methylated peptide (me0) shows the specificity of anti-rack1k271me2 antibody. H Di-methylation at the K271 residue on ectopically expressed RACK1 in HEK293T cells was determined as described in (C) except using anti-rack1k271me2 antibody (K271me2) in place of anti-pan-methyl-lysine antibody. I Di-methylation at the K271 residue on ectopically expressed RACK1 in LSD1-depleted HEK293T cells was determined as described in (H). J Identification of the LSD1-mediated regulation of methylation on endogenous RACK1 protein. Methylation on RACK1 protein in LSD1-depleted HEK293T cells was monitored by immunoprecipitating endogenous RACK1, followed by IB with the indicated antibodies. K In vitro lysine demethylase activity assay was performed using recombinant LSD1 protein and the indicated synthetic peptides. Please see the Materials and Methods section for the amino acid sequences of the peptides. Values are means SD of biological duplicates. P-values were determined by Student s t-test. Source data are available online for this figure. hardly affected by LSD1 KD (Fig 4C). Similar results were observed in experiments where the LSD1 activity was inhibited by clorgyline (Fig 4D). These results suggest that LSD1-mediated demethylation of RACK1K271me2 inhibits the binding of RACK1 to HIF-1a. Consistently, the amount of RACK1K271me2 bound to endogenous HIF-1a was significantly increased by LSD1 depletion in NCI-H596 cells (Fig 4E). Together, these results indicate that RACK1K271me2 facilitates the binding of RACK1 to HIF-1a in vivo and that the LSD1-mediated demethylation of RACK1K271me2 attenuates this interaction, thereby suppressing the ubiquitin-mediated degradation of HIF-1a. Decrease in FAD level attenuates the LSD1-mediated demethylation of RACK1 during prolonged hypoxia, causing downregulation of HIF-1a expression The robust induction of HIF-1a protein generally occurs in the early stages of hypoxia and then gradually diminishes in later stages in most cell types (Keith et al, 2012). The kinetics of HIF-1a protein expression might have important biological relevance to its function in hypoxia. We wondered whether RACK1K271me2 is dynamically regulated in different physiological conditions and hypothesized that LSD1-mediated RACK1K271me2 dynamics might be involved in the downregulation of HIF-1a protein during prolonged hypoxia. To address this question, we first examined the dynamics of RACK1K271me2 in NCI-H596 cells during prolonged hypoxia (Fig 5A). Compared to those both in normoxia and during the early stages of hypoxia (i.e., at 3 h), the RACK1K271me2 signal on the immunoprecipitated RACK1 protein gradually increased during late hypoxia (i.e., at 12 and 24 h). Importantly, the increase in RACK1K271me2 was inversely correlated with the reduction in cellular HIF-1a protein level. The late hypoxia-dependent increase in RACK1K271me2 was also observed with ectopically expressed RACK1 after 24 h of hypoxia (Fig EV4A). These observations indicate that LSD1-mediated RACK1K271me2 dynamics might indeed be responsible for the downregulation of HIF-1a during prolonged hypoxia. However, neither the transcript nor the protein level of LSD1 was prominently altered during the course of hypoxia (Fig 5A), suggesting that the enzymatic activity but not the expression level of LSD1 might determine the RACK1K271 methylation profile during prolonged hypoxia. FAD is a metabolic cofactor for LSD1 demethylase activity. We asked whether the level of FAD might be a regulating factor for LSD1-mediated demethylation of RACK1K271me2 during prolonged hypoxia. Measurement of FAD level in NCI-H596 cells during hypoxia indicated that cellular FAD level gradually decreases as hypoxia progresses in a manner that is inversely correlated with RACK1K271me2 level (Fig 5B). A similar correlative pattern of HIF-1a stability and FAD availability was also observed in Calu-6 cells (Fig EV4B and C). Investigating the reason for the decrease in FAD level during prolonged hypoxia, we observed that the expression of riboflavin kinase (RFK) and flavin adenine dinucleotide synthetase 1 (FLAD1), two enzymes essential in the FAD biosynthetic pathway, gradually diminished in the cells under prolonged hypoxic conditions, in parallel with a decrease in HIF-1a protein level (Figs 5A and EV4B). These results strongly suggest that FAD level might play a rate-limiting role in the LSD1-mediated regulation of HIF-1a stability during late hypoxia. To test this possibility, we examined the effect of RKF depletion on the hypoxic expression of HIF-1a protein. RFK depletion clearly reduced FAD level in NCI- H596 cells (Fig EV4D). In parallel, RFK KD led to a significant reduction in the hypoxia-induced HIF-1a protein level, similar to LSD1 depletion (Figs 5C and EV4E). In addition, the amount of RACK1K271me2 increased in RFK-depleted cells, reflecting the downregulation of LSD1 enzymatic activity (Fig 5D). By contrast, silencing of RACK1 increased the accumulation of HIF-1a protein in late hypoxia (Fig 5E), suggesting the involvement of RACK1- mediated degradation in the decline of HIF-1a during prolonged hypoxia. We then investigated whether the RFK-mediated FAD supply profile during hypoxia indeed parallels the HIF-mediated response patterns in hypoxic cells. Consistent with its effect on HIF- 1a protein stability, RFK depletion significantly decreased the hypoxic expression of HIF-1a target genes (Fig EV4F). Accordingly, RFK depletion caused defects in the HIF-induced phenotypes such 8 The EMBO Journal ª 2017 The Authors

9 Suk-Jin Yang et al Hypoxia responses regulated by FAD-dependent LSD1 The EMBO Journal A B C D E Figure 4. LSD1-mediated demethylation at the K271 residue on RACK1 prevents the physical association between RACK1 and HIF-1a, suppressing HIF-1a ubiquitination. A Identification of the role of K271 residue of RACK1 in the RACK1-mediated regulation of HIF-1a protein. The levels of endogenous HIF-1a protein under hypoxic conditions (1% O 2, 4 h) were determined in HEK293T cells ectopically expressing either wild-type (WT) or mutant (K271A) form of RACK1 by IB with the indicated antibodies. B Anin vivo ubiquitination assay for the RACK1K271 dependence of the ubiquitination of HIF-1a protein. HEK293T cells ectopically expressing wild-type (WT) or mutant (K/A: K271A) form of RACK1 were assessed for the ubiquitination level of HIF-1a. C Identification of the role of LSD1 and RACK1K271 in the interaction between RACK1 and HIF-1a proteins. The physical association between RACK1 and HIF-1a was monitored by immunoprecipitating the wild-type (WT) or mutant (K/A: K271A) form of RACK1 from MG132-treated (10 lm, 4 h) HEK293T cells with or without LSD1 depletion, followed by IB with the indicated antibodies. D Examination of the role of LSD1 activity and RACK1K172 in RACK1 HIF-1a interaction. Binding between RACK1 and HIF-1a proteins was examined in HEK293T cells treated with 100 lm clorgyline (16 h) as described in (C). E Examination of the role of K271 di-methylation of RACK1 in the interaction between HIF-1a and RACK1 proteins in vivo. The interaction was determined by immunoprecipitating endogenous HIF-1a from NCI-H596 cells grown in the presence of MG132 (10 lm) and desferrioxamine (1 mm) for 4 h, followed by IB with the indicated antibodies including anti-rack1k271me2 antibody. Source data are available online for this figure. ª 2017 The Authors The EMBO Journal 9

10 The EMBO Journal Hypoxia responses regulated by FAD-dependent LSD1 Suk-Jin Yang et al A B C D E F G H I Figure The EMBO Journal ª 2017 The Authors

11 Suk-Jin Yang et al Hypoxia responses regulated by FAD-dependent LSD1 The EMBO Journal Figure 5. Decrease in cellular FAD level during prolonged hypoxia causes downregulation of the LSD1-mediated RACK1K271me2 demethylation and thereby reduces the HIF-1a protein level. A The K271 di-methylation profile of RACK1 protein during the progression of hypoxia. The RACK1K271me2 level was monitored by immunoprecipitating endogenous RACK1 from NCI-H596 cells harvested at different times in hypoxia (1% O 2 ), followed by IB with the indicated antibodies. In parallel, the expression of different proteins and mrnas from the cells harvested after the indicated times under hypoxia (1% O 2 ) was also determined by IB and RT PCR, respectively. Immunoprecipitation with anti-igg antibody was performed using the cells harvested at 24 h in hypoxia. B FAD levels in NCI-H596 cells were measured at different time points in hypoxia (1% O 2 ) by determining the fluorescence of probes at Ex/Em=530/590 nm. Then, the measured FAD levels were normalized by cell numbers. Values are means SD of biological triplicates. *P < 0.05 by Student s t-test (6 h: P = , 12 h: P = , 24 h: P = ). C Examination of the RFK dependence of the HIF-1a protein expression in hypoxia. RFK- or LSD1-depleted NCI-H596 cells were cultured under hypoxia conditions (1% O 2, 6 h), and the expression of the indicated proteins and mrnas was assessed by IB and RT PCR, respectively. D Effect of RFK depletion on the level of RACK1K271me2 was monitored by immunoprecipitating endogenous RACK1 from NCI-H596 cells grown in normoxia, followed by IB with the indicated antibodies. The control IgG experiment was performed using the cells transfected with a sicontrol construct. E The effect of RACK1 silencing on the HIF-1a protein accumulation in NCI-H596 cells at different time points in hypoxia (1% O 2 ) was measured by IB with the indicated antibodies. F Effect of LSD1 loss or RFK silencing on migration and invasion of NCI-H596 cells in hypoxia (1% O 2, 24 h). Values are mean cell numbers per field SD of biological triplicates. *P < 0.01 by Student s t-test, respectively (Migration/siLSD1: P = , Migration/siRFK: P = , Invasion/siLSD1: P = , Invasion/siRFK: P = ). G Effect of silencing LSD1, RFK, or HIF-1a on tube formation of HUVEC cells in hypoxia (1% O 2, 24 h). Values are mean branch numbers per field SD for biological triplicates. *P < 0.01 by Student s t-test (sicontrol: P = , silsd1: P = , sirfk: P = , sihif-1a: P = ). H Effect of replenishing the cellular FAD pool with exogenous FAD treatment on the HIF-1a accumulation at late hypoxia. NCI-H596 cells, which were depleted of LSD1 and/or RFK in different combinations, were cultured under hypoxia (1% O 2, 24 h) with 0.01% sodium deoxycholate only ( FAD) or 0.01% sodium deoxycholate and 100 lm FAD in combination (+FAD) and analyzed for the expression of different proteins including HIF-1a and mrnas by IB and RT PCR, respectively. I Effect of exogenous FAD treatment on the RACK1271m1e2 level at late hypoxia. The very same NCI-H596 cells treated as described in (H) were analyzed for the RACK1K271me2 level by immunoprecipitating the endogenous RACK1 protein, followed by IB with antibodies for RACK1K271me2 and RACK1. Source data are available online for this figure. as glycolytic lactate production, cell motility, and induction of tube formation, to the levels comparable to the loss of LSD1 (Figs 5F and G, and EV4G, and Appendix Fig S2A and B). These results demonstrate that supply of sufficient FAD via the RFK-dependent biosynthetic pathway is critical for the accumulation of HIF-1a protein and hypoxia responses. In support of this notion, FAD supplementation experiments showed that addition of exogenous FAD to the growth medium could compensate for the deficiency in FAD level caused by RFK KD or prolonged hypoxia (1% O 2, 24 h; Fig EV4H). Further, FAD supplementation could successfully restore the HIF-1a protein accumulation for the cells in late hypoxia in an RFK-independent manner, while reducing the RACK1K271me2 level (Figs 5H and I, and EV4I). Importantly, the effect of exogenous FAD treatment was mitigated upon the KD of LSD1, implying that the FAD-dependent upregulation of HIF-1a stability was mediated by LSD1 activity. Collectively, these results indicate that cellular FAD level can act as an important rate-limiting factor for HIF-dependent hypoxia responses via modulation of LSD1-dependent RACK1 methylation dynamics and HIF-1a stability. It is also notable that attenuation of LSD1-dependent HIF-1a stability caused by the downregulation of FAD biosynthetic enzymes is directly responsible for the downregulation of HIF-1a protein level in later stages of hypoxia. HIF-1 signature in TNBC patients is positively correlated with the enzymatic activity of LSD1 and expression of FAD biosynthetic enzymes Given the role of LSD1 in mediating the stabilization of HIF-1a protein in experimental models, we wondered whether the activity of LSD1 bears any significance to HIF-1 activity in human cancers. Triple-negative breast cancer (TNBC) is strongly associated with a particularly high HIF-1 pathway signature that is associated with poor prognosis (Chen et al, 2014). We re-analyzed a publically available TNBC dataset for 579 patient samples (GSE31519), of which 114 cases had clinical information available, and confirmed the intimate association of the HIF-1 signature with poor patient outcome. However, the expression levels of LSD1 mrna in the patient group with a high HIF-1 signature were not significantly different from those in the group with a low HIF-1 signature (Appendix Fig S3). Next, we re-classified the TNBC patient samples by LSD1 activity, which was determined from the average expression of LSD1 target genes as identified by Lim et al (2010b). Overall survival analysis showed that the patient group exhibiting higher levels of LSD1 target gene expression displayed significantly poorer prognosis than the patient group with lower levels of LSD1 target gene expression (Fig 6A). This result suggests that LSD1 activity is associated with the poor prognosis of TNBC patients. Furthermore, the average expression of LSD1 signature genes from all 579 TNBC samples was strongly correlated with the HIF-1 pathway signature (R = 0.452; Fig 6B), suggesting that LSD1 activity is significantly involved in regulating the HIF-1 pathway in TNBC, thereby affecting cancer progression. We also found that the expression of RFK and FLAD1 was highly correlated not only with the expression of the LSD1 signature but also with HIF activity (Fig 6C and D), implying that cellular FAD production affects the expression of HIF target genes by modulating LSD1 activity. In support of this possibility, the expression of FAD biosynthetic genes was significantly correlated with the poor prognosis of TNBC patients (Fig 6E). These results collectively suggest that LSD1 may have significant clinical relevance in TNBC patients by promoting FAD-dependent upregulation of the HIF pathway. Discussion In this study, we present a mechanistic insight into how the FADdependent lysine demethylase LSD1 regulates HIF-induced responses, such as the upregulation of glycolysis in hypoxia. Our ª 2017 The Authors The EMBO Journal 11

12 The EMBO Journal Hypoxia responses regulated by FAD-dependent LSD1 Suk-Jin Yang et al A B C D E F Figure 6. findings showed that (i) the lysine demethylase activity of LSD1 is critical for HIF-1a protein accumulation by preventing its oxygenindependent degradation by the RACK1-Hsp90 pathway, (ii) LSD1 is responsible for the demethylation of K271me2 on RACK1 (a component of the HIF-1a ubiquitination machinery), resulting in the inhibition of the physical interaction of RACK1 with HIF-1a (Fig 6F), 12 The EMBO Journal ª 2017 The Authors

13 Suk-Jin Yang et al Hypoxia responses regulated by FAD-dependent LSD1 The EMBO Journal Figure 6. The HIF-1 signature is positively correlated with the expression of LSD1 target genes along with the expression of FAD biosynthetic enzymes in TNBC. A Kaplan Meier graphs showing a significant association of high LSD1 signature expressing group (top 30%) with shorter survival as compared to those of low LSD1 signature group (bottom 30%) in a cohort of 114 TNBC patients. The log-rank test P-value is shown. B Positive correlation between the expression of LSD1 target genes (LSD1 signature) and HIF-1 signature in a cohort of 579 TNBC patients. P-value was calculated by the log-rank test. R: correlation coefficient. C Positive correlation between the expression of FAD biosynthetic enzymes, RFK and FLAD, and LSD1 signature in a cohort of 579 TNBC patients. P-value was calculated by the log-rank test. R: correlation coefficient. D Positive correlation between the expression of FAD biosynthetic enzymes, RFK and FLAD, and HIF-1 signature in a cohort of 579 TNBC patients. P-value was calculated by the log-rank test. R: correlation coefficient. E Kaplan Meier graphs showing a significant association of high RFK/FLAD1 expressing group (top 30%) with shorter survival as compared to those of low expression group (bottom 30%) in a cohort of 114 TNBC patients. The log-rank test P-value is shown. F Schematic model for the LSD1-mediated regulation of HIF-1a protein stability by controlling RACK1 protein methylation in hypoxia. (iii) a change in the level of FAD, a metabolic cofactor for LSD1 demethylase activity, plays a rate-limiting role in HIF-1a stability during prolonged hypoxia, and (iv) LSD1 activity and the expression of FAD biosynthesis enzymes are strongly correlated with HIF-1 signature gene expression in human cancer. The growing list of non-histone substrates for LSD1 raises its importance beyond the role in chromatin modification. Our finding that LSD1 demethylates K271me2 of RACK1 and thereby protects HIF-1a from the RACK1-mediated degradation reinforces this point. Previously, Qin et al reported that HIF-1a might be regulated by LSD1 in pancreatic cancers (Qin et al, 2014). They suggested LSD1 as a recruiting factor for histone lysine deacetylases to HIF-1a, which consequently suppresses acetylation-dependent HIF-1a degradation. However, our analyses with a catalytically dead LSD1 mutant and different LSD1 inhibitors (Figs 1 and 2, and EV1 and EV2) clearly indicate a more direct role of LSD1 as an enzyme in the regulation of HIF-1a protein stability, because the lysine demethylase activity of LSD1 was required for increasing HIF-1a stability. Supporting this finding, regulation of HIF-1a protein methylation by LSD1 and Set7/9 lysine methyltransferase has been recently reported (Kim et al, 2016). By contrast, our cellular and in vitro assays showed that LSD1 demethylates the di-methylation of K271 residue on RACK1 (Fig 3). Furthermore, K271A mutation on RACK1 rendered the physical association between RACK1 and HIF-1a irresponsive to the activity of LSD1 (Fig 4). These results reveal a novel mechanism of the non-chromatic action of LSD1; that is, LSD1-mediated demethylation of K271 on RACK1 inhibits the binding of RACK1 to HIF-1a and consequently suppresses RACK1- mediated HIF-1a degradation. The level of HIF-2a, another target of RACK1-dependent degradation, was also affected by LSD1 (Fig EV2), indicating that the non-histone lysine demethylase function of LSD1 plays a key regulatory role in the RACK1-mediated ubiquitination pathway that is common for the degradation of both HIF-a subunits. Methylation of lysine residues on histones or non-histone proteins such as p53 produces a binding motif for interacting proteins (Huang et al, 2007; Musselman et al, 2012). RACK1, being composed of seven WD domains, is a multifaceted scaffolding protein that physically interacts with several proteins including HIF-1a (Adams et al, 2011). The lysine 271 residue on RACK1 is located at the region extended from the WD6 domain, which is the minimal region for cellular HIF-1a binding (Liu et al, 2007). Based on our finding that the overexpression of wild-type RACK1, but not RACK1(K271A) mutant, decreased HIF-1a protein level (Fig 4), we postulate that K271 residue participates in the formation of the binding surface for HIF-1a and that modification of K271 can exert crucial effects on the interaction between RACK1 and HIF-1a. RACK1 and Hsp90 competitively bind to the PAS-A domain located at the N-terminal region of HIF-1a (Liu & Semenza, 2007). Although the PAS-A domain does not display structural similarity to defined methyl-lysine binders, our findings suggest that RACK1 with K271me2 acquires a stronger HIF-1a binding affinity than Hsp90, balancing subsequent reactions toward the proteasomal degradation of HIF-1a. Future identification of a lysine methyltransferase responsible for RACK1K271 methylation will increase our understanding of the physiological role of RACK1 as a regulator of HIF-mediated responses induced in different microenvironments. The enzymatic activity of LSD1 requires FAD, a metabolite derived from riboflavin and ATP. The LSD1-mediated enzymatic reaction converts FAD to a reduced form, FADH 2 (Barile et al, 2000). In this study, we found that the cellular amount of FAD decreases in prolonged hypoxia, as the expression of the ratelimiting enzymes of FAD biosynthesis, such as RFK and FLAD1, was downregulated with the progression of hypoxia (Fig 5). Further, we observed the RACK1-dependent diminution of HIF-1a protein in prolonged hypoxia, which was correlated with the decrease in FAD and increase in RACK1K271me2 in the absence of a prominent change in LSD1 expression at both mrna and protein levels. These findings indicate that cellular FAD level and FAD-dependent LSD1 activity regulating RACK1K271me2 might be the major determinants of HIF-1a stability during prolonged hypoxia. In line with these findings, ablation of RFK expression reduced not only the cellular amount of FAD but also HIF-1a protein level, consequently downregulating HIF-1 target gene expression. Furthermore, either RACK1 depletion or exogenously supplied FAD facilitated the induction of HIF-1a protein in FAD-depleted conditions, such as prolonged hypoxia. It is notable that the HIF-1a level in RACK1-depleted cells at late hypoxia was still lower than that at early hypoxia. Considering the previous findings that the upregulation of PHD3 was implicated in regulating the HIF-1a protein stability under chronic hypoxia in some models (Appelhoff et al, 2004; Ginouves et al, 2008), we speculate that the induction of PHD3 activity and the suppression of LSD1 activity due to decrease in cellular FAD level might function in combination to downregulate the HIF-1a protein stability during prolonged hypoxia. FAD availability in cells can also be modulated by the conversion between FAD and FADH 2 through oxidoreductive reactions involved in key metabolic pathways, such as fatty acid b-oxidation and tricarboxylic acid (TCA) cycle coupled with oxidative phosphorylation. ª 2017 The Authors The EMBO Journal 13