HIF-1α induces MXI1 by alternate promoter usage in human neuroblastoma cells

E XP E RI ME N TA L C E L L R E S EA RC H 315 ( 2 0 0 9 ) 1924 – 193 6

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y e x c r

Research Article

HIF-1α induces MXI1 by alternate promoter usage in human neuroblastoma cells Tobias Löfstedt a , Erik Fredlund a , Rosa Noguera b , Samuel Navarro b , Linda Holmquist-Mengelbiera , Siv Beckman a , Sven Påhlman a , Håkan Axelson a,⁎ a

Center for Molecular Pathology, Department of Laboratory Medicine, Lund University, University Hospital MAS, Entrance 78, S-205 02 Malmö, Sweden b Department of Pathology, University of Valencia, Medical School, Valencia, Avda Blasco Ibañes 17, 46010 Valencia, Spain

A R T I C L E I N F O R M AT I O N

AB ST R AC T

Article Chronology:

Adaptation to low oxygen conditions is essential for maintaining homeostasis and viability in

Received 4 December 2008

oxygen-consuming multi-cellular tissues, including solid tumors. Central in these processes are the

Revised version received

hypoxia-inducible transcription factors, HIF-1 and HIF-2, controlling genes involved in e.g. glucose

12 February 2009

metabolism and neovascularization. Tumor hypoxia and HIF expression have also been associated

Accepted 12 February 2009

with a dedifferentiated phenotype and increased aggressiveness. In this report we show that the

Available online 27 February 2009

MAX interactor-1 (MXI1) gene is directly regulated by HIF proteins in neuroblastoma and breast cancer cells. HIF-binding and transactivation were detected within MXI1 gene regulatory

Keywords:

sequences in the vicinity of the MXI1-0 promoter, leading to rapid induction of the alternate

MXI1

MXI1-0 isoform followed by a long-term induction of both the MXI1-0 and MXI1 isoforms.

Hypoxia

Importantly, knock-down of MXI1 had limited effect on MYC/MYCN activity under hypoxia, an

HIF-1

observation that might be related to the different functional attributes of the two MXI1 isoforms.

Hypoxia responsive elements (HRE)

© 2009 Elsevier Inc. All rights reserved.

Neuroblastoma MYCN

Introduction Hypoxia frequently arises in solid tumors as a result of oxygenconsuming high cellular proliferation, and improper neovascularization causing insufficient delivery of new oxygen. Through an adaptive response, primarily mediated by the hypoxia-inducible transcription factors, HIF-1 and HIF-2, cancer cells can survive and proliferate under hypoxic conditions [1,2]. HIF proteins are composed of an α-subunit, HIF-1α or HIF-2α, and the constitutively present dimerization partner ARNT/HIF-1β [2]. At normoxia, HIF-α proteins become hydroxylated by prolyl hydroxylase domain-containing proteins (PHDs) [3–5], leading to recognition

⁎ Corresponding author. Fax: +46 40 337063. E-mail address: [email protected] (H. Axelson). 0014-4827/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2009.02.015

and ubiquitinylation by the von Hippel-Lindau (VHL)–E3 ligase complex [5, 6], and subsequent degradation of the HIF-α subunits via the proteasome [7–9]. The transcriptional activity of HIF-α proteins is inhibited under normoxia via hydroxylation of an asparagine residue by the oxygen-dependent factor inhibiting HIF (FIH) [10,11]. At hypoxia (generally considered less than 2% O2), HIF-α proteins become stabilized, heterodimerize with ARNT and regulate target genes involved in the adaptation to hypoxic conditions via binding to genomic hypoxia-response elements (HREs) [2,12]. In recent reports, we analyzed the hypoxic response in cells of the pediatric tumor neuroblastoma using the microarray

E XP E RI ME N TA L C E L L R E S EA RC H 315 ( 2 0 09 ) 19 24 – 193 6

technique, and found several significantly hypoxia-induced genes [13,14]. Many of these genes were controlled by both HIF-1 and HIF-2, however in an oxygen- and temporally regulated manner. In the list of hypoxia-responsive genes we identified the MAX interactor-1 (MXI1) gene. MXI1 is a member of the MYC– MAX–MAD network of transcription factors, and due to its high structural and functional similarity with MAD proteins, MXI1 is considered to act as an antagonist of the oncogene MYC [15,16]. MXI1 exerts this function through heterodimerization with the obligate MYC partner MAX and thereby transcriptionally represses genes otherwise activated by MYC–MAX dimers [15,16]. However, recent work has indicated that MXI1 can regulate genes distinct from those controlled by MYC [17]. Furthermore, two major alternatively transcribed isoforms of MXI1, conserved between mouse and human, have been described. In humans the primary transcript is termed MXI1 (in this text referred to as MXI1-1) and the alternate MXI1-0, in mouse the corresponding transcripts are termed Mxi1-SRβ and Mxi1-SRα, respectively [18,19]. The functional differences between these two isoforms remain unclear, but recent data indicate that they may differ in their capacity to modulate MYC function [20]. MXI1 has been suggested to be a potential tumor suppressor and loss of mxi1 in mice also leads to increased susceptibility to tumorigenesis following carcinogenic exposure [21], while overexpression of MXI1 can result in decreased growth of prostate tumor and glioblastoma cells [22,23]. MXI1 is located on chromosome 10q24–25 [24], a genomic region often exhibiting loss of heterozygosity in some human cancers [25–27], but according to some reports the MXI1 locus appears to be an unlikely major target of these allelic losses [27,28]. MXI1 has also been found to harbor mutations in prostate tumors [25,29], but these mutations appear to be very rare and their general role in human cancer is currently debated [30–33]. Activation of HIF-1α has been shown to counteract MYC by downregulating MYC-activated genes leading to cell cycle arrest [34,35]. In contrast, HIF-2α might potentiate MYC function and thereby promote growth [34]. Since aggressive neuroblastoma growth is highly associated with MYCN-amplification [36], we investigated MXI1 induction under hypoxic conditions in neuroblastoma cells. Previous reports have demonstrated induction of MXI1 by hypoxia in several different cell types [37–41], indicating a general and an important function of MXI1 in the process of adaptation to low oxygen. A direct role of HIF-1 in the hypoxic induction of MXI1 was recently shown [38,42], but the precise mechanisms by which HIF-1 induces MXI1 have not been addressed. In this report we show that hypoxia leads to upregulation of MXI1 mRNA and protein in neuroblastoma and breast cancer cells, and that HIF proteins are essential for this induction via binding to HREs within MXI1 gene regulatory sequences. Although MYCN levels, as well as MYC/MYCN target genes were downregulated at hypoxia, knock-down of MXI1 by siRNA only resulted in limited effects on MYC/MYCN target gene expression in hypoxic neuroblastoma cells. Importantly, we show differential hypoxic regulation of the two major isoforms, MXI1-1 and MXI1-0, which could have impact on the net MXI1 function under low oxygen. These findings highlight a novel regulatory mechanism by which HIF proteins can regulate the function of primary target genes and provide further evidences for the importance of considering the temporal aspects of the hypoxic response.

1925

Material and methods Cell culture SK-N-BE(2)c, SK-N-SH, KCN-69n, IMR-32, LA-N-5 and SK-N-FI neuroblastoma cells, and MCF-7 and T47D breast cancer cells were maintained in conventional cell culture media under standard growth conditions. At normoxia (i.e. atmospheric pressure around 21% O2), cells were kept in a humidified chamber at 37 °C in 5% CO2 and 95% air. Hypoxia (1% O2) was created in a Hypoxia workstation connected to a Ruskinn gas mixer module (Ruskinn Technology). In a set of experiments actinomycin D (Sigma) was added to a final concentration of 5 μg/ml medium.

Real-Time Quantitative PCR (QPCR) Total RNA was extracted using QIAshredder spin columns and RNeasy kit according to the manufacturer's instructions (Qiagen Sciences). Samples were DNase-treated and washed before cDNA was generated using random hexamers and Superscript II RT-enzyme (Invitrogen). Obtained cDNA was used as template in quantitative PCR reactions together with SYBR Green PCR Master Mix (Applied Biosystems), and samples were run on a 7300 Real Time PCR System (Applied Biosystems). For relative quantification of mRNA expression levels, the comparative Ct method was used [43], and data was normalized to the expression of three reference genes not regulated by hypoxia (SDHA, YWHAZ and UBC). QPCR primers were designed using Primer Express software (Applied Biosystems) and sequences are given in Supplemental Table 1.

Tumor material, immunohistochemistry and Western blotting Cells were seeded on cover slips 16–18 h before incubation at normoxia or hypoxia for 24 h. Thereafter, cells were fixed in 4% paraformaldehyde for 10 min and mounted in PVA-DABCO. For immunodetection of MXI1 protein, the polyclonal anti-Mad2 (G16) antibody (Santa Cruz Biotechnology) was used followed by incubation with fluorescently labeled secondary antibodies, Alexa488 (Molecular Probes). Sections of formalin-fixed, paraffin embedded human breast carcinoma in situ (DCIS) specimens, were analyzed by HIF-1α immunohistochemistry using an anti-HIF-1α antibody (Abcam) as described [44]. All analyzed lesions were invasive ductal carcinomas of histological grade III and frequently exhibited central necrosis. DCIS specimens were also stained for MXI1 using the anti-Mad2 (G16) antibody (Santa Cruz). After antigen retrieval in high pH buffer, immunoreactivity was detected using the Dako REAL EnVision Detection System in a DAKO TechMate 500 (DAKO A/S, Glostrup, Denmark). A tissue microarray (TMA) generated from routinely fixed and embedded tissue samples of 93 individual cases of neuroblastoma was used (described in detail elsewhere [14]; ethical approval no. 59CI8ABR2002). The TMA sections were analyzed by HIF-1α and MXI1 immunohistochemistry as described above. Immunoreactivity was independently scored by two pathologists and classified according to range, i.e. fractions of positive cells (0: 0–10%, 1: 11–25%, 2: 26–75%, 3: 76–100%) and general intensity of positive

1926

E XP E RI ME N TA L C E L L R E S EA RC H 315 ( 2 0 0 9 ) 1924 – 193 6

cells (0: none, 1: mild, 2: moderate, 3: intense). As a negative control, adjacent tissue sections were processed by replacing the primary antibody with non-immune rabbit IgG (code X0936), or a

non-immune mouse monoclonal antibody (code X0931, both from DAKO). Western blotting was performed as previously described [14].

Fig. 1 – The MXI1 gene is induced by hypoxia in neuroblastoma cells. (A) Genomic organization of MXI1 showing the alternative first exons (0 and 1, respectively) of the expressed isoforms MXI1-0 and MXI1-1, and common exons 2–6. Resulting mRNA species are given below. (B–D QPCR experiments of SK-N-BE(2)c cells, grown at 21 or 1% O2 for 0, 4, 8, 24 and 72 h using primers specific for the MXI1-1 (B) and MXI1-0 (C) isoforms and common exon 6 (D). Data was normalized to the expression of three reference genes (SDHA, YWHAZ, and UBC) not affected by hypoxia (data not shown) and related to time-point zero (0) set to 1. Error bars indicate ± SD within triplicates. (E–G) Analysis of MXI1-0 mRNA levels using QPCR, in KCN-69n (E), IMR-32 (F) and SK-N-FI (G) neuroblastoma cells cultured at hypoxia for 8 or 48 h. For comparison, mRNA levels at 21% O2, 8 h, are shown. H: Immunohistochemical stainings of SK-N-BE(2)c cells (upper panels) and KCN-69n cells (lower panels) after 24 h at 21 or 1% oxygen as indicated, using antibodies targeting MXI1 (magnification × 1000).

E XP E RI ME N TA L C E L L R E S EA RC H 315 ( 2 0 09 ) 19 24 – 193 6

1927

Chromatin immunoprecipitation (ChIP)

Transfection and siRNA

SK-N-BE(2)c cells were cultured at hypoxia or normoxia for 4 h before DNA–protein cross-linking in PBS + 1% formaldehyde at room temperature for 10 min. Cells were then washed, lysed and the lysate was immunoprecipitated as described [45], using monoclonal anti-HIF-1α antibodies (Novus) or control anti-IgG antibodies (Abcam). Precipitated DNA was purified and used as template for 35 cycles of PCR together with primers flanking putative HIFbinding sites in MXI1 regulatory sequences (Supplemental Table 2).

Neuroblastoma cells were seeded in 6- or 24-well culture plates (Costar, Corning Incorporated) at ∼50% confluency approximately 16– 18 h before transfections, which were carried out in Opti-MEM I Reduced Serum Medium and Lipofectamine-2000 Reagent (Invitrogen) for 5–6 h. Thereafter, transfection mix was diluted 1:1 with serum containing medium (in order to maintain cell viability without compromising transfection efficiency), and cells were left an additional 16–18 h prior to incubation at normoxia or hypoxia. Annealed and

Fig. 2 – Hypoxic upregulation of MXI1 is dependent on HIF proteins. (A) Actinomycin D (AD) abrogates hypoxic induction of MXI1 in SK-N-BE(2)c cells, as determined by QPCR analysis. Results of MXI1 exon 6 amplicon are illustrated and similar results were found using MXI1-0-specific primers (data not shown). (B) Western blot demonstrating functionality and specificity of siRNAs against HIF proteins in SK-N-BE(2)c cells grown for 24 h at hypoxia. The scrambled control siRNA had no significant effect on either HIF α-subunit. (C–H) QPCR experiments of SK-N-BE(2)c cells cultured for 4 h at 21 or 1% O2, using selective siRNA treatment against HIF-1α or HIF-2α. Effects on the expression of HIF-1α (C) and HIF-2α (D) mRNAs by cognate siRNA treatment were investigated, as well as expression of the known HIF target gene VEGF (E), and the expression of MXI1-0 (F), MXI1 exon 6 (G) and MXI1-1 (H). (I–J) HIF siRNA analysis demonstrating HIF-independence of MXI1-1 induction (I), but involvement of both HIFs for MXI1-0 induction (J) in cells cultured at prolonged hypoxia (48 h). Error bars show ± SD within triplicates.

1928

E XP E RI ME N TA L C E L L R E S EA RC H 315 ( 2 0 0 9 ) 1924 – 193 6

HPLC-purified small inhibitory RNA (siRNA) from Ambion (Ambion Inc.) against either HIF-1α (sense: CUG AUG ACC AGC AAC UUG AdTdT and antisense: UCA AGU UGC UGG UCA UCA GdTdT) or HIF-2α (sense: CAG CAU CUU UGA UAG CAG UdTdT and antisense: ACU GCU AUC AAA GAU GCU GdTdT) were employed in transfections at a final concentration of 50 nM. For control, the scrambled HIF-1α siRNA sequence was used (sense: UCA GCA CAU GGA UUC GCA AdTdT and antisense: UUG CGA AUC CAU GUG CUG AdTdT), which did not specifically target any tested gene. The MXI1 siRNA sequences was predesigned by Ambion, (sense: CCC UUC CUG AGC UUU AUG GdTdT and antisense: CCA UAA AGC UCA GGA AGG GdTdT) and was directed against exons common for both analyzed MXI1 isoforms.

DNA constructs and Luciferase assay For Luciferase experiments, SK-N-BE(2)c or SK-N-SH cells were transfected as described above, using 100–600 ng of Luciferase-

expressing plasmids per transfection in 24- or 6-well culture plates. MXI1 genomic fragments were amplified by PCR; MXI1B promoter forward: ACT ACG CGT TAG CTT GGC ACA TTC GTC GTT C and reverse: ACT CTC GAG ATG GGA GTG TGT GTC GGT CT; MXI1 IVS (HRE 1,2,3,4, nt +1560 to +3326) forward: ACT ACG CGT GTG CAG CGT ATC and reverse: ACT CTC GAG CTG AAT GAT GTA AGC GTT TCG G, before cloned into the pGL3Basic vector (Promega). Deletion constructs of the MXI1 IVS reporter were created using following primers; (HRE 1,2,3) forward: same as full-length MXI1 IVS; (HREs 2,3,4, nt +1774 to + 3326) forward: ACT ACG CGT GTG TCA GAT CTT GCA ACC TTC CC; (HRE 3,4, nt +2215 to +3326) forward: ACT ACG CGT GGC TAC CAG TTC TCT GGA GTC TG; and (HRE 4, nt + 2837 to +3326) forward: ACT ACG CGT CCC AGA TTT CCC CGA GAC CTT. Reverse primers were: ACT AAG CTT CTG AAT GAT GTA AGC GTT TCG, except for the (HRE 1,2,3) reverse primer: ACT AAG CTT TTC CGT ACC CAG AGA CCT AGA. The 3 × EPO HRE construct has previously been

Fig. 3 – HIF-1 binds and activates MXI1 via HREs located in gene regulatory sequences. (A) Hypoxia induces activity of a genomic intervening sequence (IVS) located 3′ of the MXI1-0 first exon (dashed box in diagram). The MXI1 IVS fragment (∼1.8 kbp) was cloned in front of the pGL3Basic Luciferase gene and SK-N-BE(2)c cells were transiently transfected with 300 or 600 ng of the construct. After 6 h of transfection, cells were switched to serum containing medium and left overnight before incubation at hypoxia or normoxia for 24 h. Luciferase activity was measured and normalized against the levels of co-transfected CMV-Renilla Luciferase, reflecting efficiency of transfection. Empty Luciferase vector was used as negative control, and a construct containing three copies of the EPO HRE (3 × HRE) was used as positive control. (B) Hypoxic induction of the MXI1 IVS is HIF-1 dependent. SK-N-BE(2)c cells were transfected with 300 or 600 ng of the MXI1 IVS together with siRNA targeting HIF-1α or control siRNA (50 nM). (C) HIF-1 binds the MXI1 IVS in vivo. ChIP experiments in SK-N-BE(2)c cells grown at 21 or 1% O2 for 4 h, demonstrating HIF-1-binding at two putative HREs within the MXI1 IVS, designated +2140 HRE (2) and +2819 HRE (3), respectively. Equal genomic DNA input amounts were determined for the 21 and 1% O2 samples (data not shown). (D) The IVS HREs are required for HIF-1-induced activation of MXI1. K-N-BE(2)c cells were separately transfected with 300 ng of different MXI1 IVS deletion constructs containing potential HREs as indicated, together with 500 ng CMV-driven constitutively active HIF-1α or control CMV plasmids. Cells were harvested after 48 h followed by measurements of Luciferase activities. All data is representative of at least two independent experimental setups yielding similar results. For Luciferase assays, error bars indicate ± SD of at least triplicate transfections.

E XP E RI ME N TA L C E L L R E S EA RC H 315 ( 2 0 09 ) 19 24 – 193 6

described [8]. In a set of experiments, a CMV-driven plasmid expressing constitutively active HIF-1α was used (kindly provided by Dr. Lorenz Poellinger, Karolinska Institute, Stockholm, Sweden). Specific siRNAs were included in transfections as indicated. For control of transfection efficiency, 50–100 ng of a CMV-Renilla Luciferase vector (Promega) was included in all samples. After 16–18 h of post-transfectional recovery, cells were placed in normoxia or hypoxia for 24–48 h. Cells were thereafter harvested and Luciferase activities were determined using the Dual-Luciferase Reporter system according to the manufacturer's guidelines (Promega).

Microarray For gene expression microarrays, RNA from SK-N-BE(2)c and SKN-SH cells transfected with siRNA against MXI1 or control siRNA and grown at + 1 % O2 for 24 h, was isolated. Array production, sample labeling, hybridization and scanning were performed essentially as described [46]. For each sample, 5 μg of total RNA was labeled with Cy3 and 5 μg with Cy5. Each MXI1 siRNAtransfected sample was then hybridized to the corresponding control siRNA sample in dye-swap duplicates, giving a total of 12 hybridizations. TIFF images were analyzed using the Gene Pix Pro 4.0 software (Axon Instruments, Foster City, CA) and the quantified intensity data was loaded into the BioArray Software Environment (BASE) [47]. Within BASE, background-corrected Cy3 and Cy5 intensities were calculated using the median spot pixel intensity and median local background pixel intensity. Fluorescence ratios were calculated as MXI1 siRNA intensity over control siRNA intensity, irrespective of Cy-dye label. After quality filtering, the dye-swap assay pairs were merged by averaging ratios over each array oligomer, hereafter called reporter. The log (2) ratios for each merged assay were normalized and corrected for intensity-based adjustment [48]. Gene regulation was analyzed using an R-implementation of rank product analysis [49]. For the separate analysis of the two transfected cell lines, one missing value in the three replicates was allowed and substituted with the mean of the other two log(2) ratios. For SKN-BE(2)c samples this rendered a data set of 11 109 reporters (number of missing values = 1635), and for SK-N-SH samples a data set of 11,713 reporters (number of missing values = 320). For the second round of analysis, all six microarray assays were analyzed as one class, using the union of the previously mentioned SK-N-BE(2)c and SK-N-SH data sets (n = 10,964). The rank product analysis gave false discovery rates (FDR or q) pertaining to both up- and downregulation for each gene, and this measure was used to create ranked gene lists based on rank product analysis. The ranked gene lists were used for correlation analyses to known gene signatures according to the gene set enrichment analysis method [50], using a gene signature (gene set) relating to MYC-based gene regulation. The MYC gene set was obtained from the MYC cancer gene database [51] as previously described [52]. The gene set enrichment analysis was performed using the GseaPreranked tool, and normalized enrichment score (NES), FDR (q) were considered for each ranked gene list. The microarray raw data are available in MIAME compliant format from the ArrayExpress database (http://www.ebi.ac.uk/arrayexpress/) with accession number E-MEXP-2033.

1929

Results MXI1 is induced by hypoxia in neuroblastoma cells Using QPCR primers specific for MXI1-1 and MXI1-0 isoforms, and MXI1 exon 6, common to both isoforms (Fig. 1A), we detected induction of the MXI1-1 (Fig.1B) and the MXI1-0 isoforms (Fig.1C) in SK-N-BE(2)c neuroblastoma cells cultured at hypoxia (1% oxygen) up to 72 h. The upregulation of MXI1-0 was faster than that of MXI1-1 and resembled the induction of MXI1 exon 6 (Figs. 1C and D), suggesting that MXI1-0 is the predominant transcript in hypoxic SK-N-BE(2)c cells, which was confirmed by direct comparison of mRNA levels (data not shown). The fast and sustained hypoxic induction of MXI1-0 observed in SK-N-BE(2)c cells was also detected in KCN-69n (Fig. 1E), IMR-32 (Fig. 1F), and SK-N-FI (Fig. 1G) neuroblastoma cells. In addition, hypoxia markedly increased MXI1 protein expression in the two neuroblastoma cell lines investigated (Fig. 1H). Notably, high levels of nuclear MXI1 were observed at 1% oxygen when compared to cells grown at 21% oxygen.

Upregulation of MXI1 under hypoxia requires HIF proteins By inhibiting the transcriptional machinery via actinomycin D (AD) treatment, the early hypoxic induction of MXI1 (exon 6) in SK-N-BE(2)c cells was completely abrogated (Fig. 2A). Similar results were obtained using MXI1-0 isoform-specific primers (data not shown), indicating direct transcriptional regulation of MXI1 under hypoxia. To test for potential involvement of HIF proteins, siRNA against either HIF-α subunit was employed, and specific and efficient HIF knock-down was confirmed (Figs. 2B–D). As previously shown [14], HIF-1α in contrast to HIF-2α, was essential for full activation of classical HIF target genes such as VEGF (Fig. 2E) under acute hypoxia. When examining MXI1 levels in HIF siRNAtreated cells, induction of MXI1-0 as opposed to MXI1-1 (Figs. 2F– H) displayed a strong dependence of HIF-1α protein after 4 h of hypoxia. However, after 48 h of hypoxic culture both MXI1-1 and MXI1-0 (Figs. 2I, J) isoforms were substantially upregulated, but interestingly, only induction of the MXI1-0 isoform was affected by

Fig. 4 – MXI1 protein correlates with HIF-1α in neuroblastoma specimens. MXI1 and HIF-1α immunoreactivities, measured on a neuroblastoma tissue microarray, correlate positively as described by staining intensity of positive cells (ρ = 0.31, p = 0.004, n = 82, Spearman correlation).

1930

E XP E RI ME N TA L C E L L R E S EA RC H 315 ( 2 0 0 9 ) 1924 – 193 6

HIF knock-down. In addition, and in accordance with previous findings on HIF target gene regulation [14], HIF-2α was also involved in the upregulation of MXI1-0 expression under prolonged hypoxia (Fig. 2J).

MXI1 is a direct HIF target gene Since MXI1-0 appeared to be the primary HIF-induced MXI1 transcript in neuroblastoma cells, we examined the MXI1-0 promoter for presence of putative hypoxia-response elements (HREs). Constructs containing 2.5 kbp of the proximal MXI1-0 promoter were however not induced by hypoxia (Supplemental Fig. 1A). Accordingly, chromatin immunoprecipitation (ChIP) on the three existing putative HREs based on the determined consensus HIF-binding site [12] within the proximal MXI1-0

promoter did not show any specific or consistent HIF-1 binding, while binding to the VEGF promoter HRE was detected under these conditions (Supplemental Fig. 1B). HIF-binding sites have previously been reported within intron sequences of a number of hypoxic target genes (reviewed in [12]), and several potential HREs (Supplemental Table 2) were found 3′ of the MXI1-0 exon. We selected a 1.8 kbp DNA fragment, termed MXI1 intervening sequence (IVS), from this genomic region and introduced it in front of a Luciferase reporter gene. As shown in Fig. 3A, the MXI1 IVS construct was clearly induced in SK-N-BE(2)c cells cultured at 1% oxygen for 24 h. When co-transfecting siRNA targeting HIF-1α, the hypoxic reporter gene activation was completely inhibited (Fig. 3B). The MXI1 IVS genomic fragment contains four putative HIF-binding sites, here designated HREs 1–4. Performing ChIP assays on these sequences, and on potential HREs located outside

Fig. 5 – MXI1 is induced by hypoxia in breast cancer cells. (A, B) QPCR analysis showing MXI1-0 upregulation at 1% O2 in MCF-7 (A) and T47D (B) breast cancer cells after 4 and 72 h incubation. The MXI1-1 isoform is also induced by hypoxia, but only at the later time-point and with lower magnitude, in accordance with the overall MXI1 pattern seen in hypoxic neuroblastoma cells. Levels at 21% O2, 4 h, are presented for comparison. (C) Immunohistochemical stainings of MXI1 in breast cancer cells after 24 h of hypoxia or normoxia, as indicated (magnification × 1000). (D) MXI1 protein increases in HIF-1α-high regions of breast ductal carcinoma in situ (DCIS). IHC evaluation of a histological grade III DCIS specimen using antibodies against HIF-1α and MXI1, respectively. Arrows indicate positive nuclear staining.

E XP E RI ME N TA L C E L L R E S EA RC H 315 ( 2 0 09 ) 19 24 – 193 6

of the isolated MXI1 fragment (data not shown), revealed specific HIF-1-binding at two sites within the MXI1 IVS in hypoxic SK-N-BE (2)c cells (Fig. 3C). Experiments with MXI1 IVS deletion constructs showed that the identified HIF-binding sites, termed HRE 2 and HRE 3, were both required for full reporter gene expression induced by co-transfected HIF-1α (Fig. 3D). Interestingly, although HRE 4 did not exhibit any consistent or specific HIF-1 binding and was not involved in transcriptional MXI1 enhancement by HIF-1, DNA sequences surrounding this site appeared to be important for basal activity of the investigated genomic region (Fig. 3D).

MXI1 correlates with HIF-1a in neuroblastoma specimens and hypoxia increases MXI1 expression in breast cancer cells To test whether the HIF-1α-dependent induction of MXI1 in cultured hypoxic neuroblastoma cells is reflected also in a clinical neuroblastoma material, we immunostained a tissue microarray constructed from 93 individual neuroblastoma cases with antibodies directed against HIF-1α and MXI1, respectively. Although the majority of tumor section cores stained negative for MXI1 (47 out of 82 that could be scored), there was a significant correlation between high MXI1 protein and HIF-1α staining in sections that were positive for MXI1 (Fig. 4), suggesting that HIF-1α regulates MXI1 also in vivo. Hypoxia substantially augmented MXI1-0 expression also in MCF-7 and T47D breast carcinoma cells, both at acute (4 h) and prolonged (72 h) hypoxia. Similar to the pattern observed in neuroblastoma cells, the MXI1-1 isoform was only induced with low magnitude after 4 h of culture at 1% O2 (Figs. 5A, B). The upregulation of MXI1 mRNA was accompanied by an increased accumulation nuclear MXI1 protein in hypoxic breast cancer cells (Fig. 5C). In addition, when examining specimens of breast ductal carcinoma in situ that frequently contain necrotic areas with surrounding hypoxic/HIF-expressing region of cells (Fig. 5D and [44]), relatively strong MXI1 staining could be detected in the HIF-1 expressing perinecrotic zone of cells (Fig. 5D). Together, these experiments show that hypoxia induces the MXI1-0 isoform also in human breast cancer cells and that this induction most likely contributes to elevated levels of MXI protein detected in hypoxia treated breast cancer cells and in hypoxic regions of primary breast cancer.

Knock-down of MXI1 moderately affects MYC/MYCN activity in hypoxic neuroblastoma cells To investigate the role of MXI1 induction at hypoxia, we performed microarrays using RNA from SK-N-BE(2)c (MYCNamplified) and SK-N-SH (non-MYCN-amplified) neuroblastoma cells, transfected with siRNAs targeting total MXI1 (si-Mx) or control (si-C), and thereafter cultured under hypoxic conditions for 24 h. These cells were chosen in order to establish a putative role of hypoxia-regulated MXI1 in a background of high and low MYCN expression, respectively. Treatment with siRNA against MXI1 efficiently downregulated both isoforms in investigated cell lines, as exemplified by MXI1-0 (Fig. 6A). The efficacy of the siRNA transfection was confirmed at the protein level using immunohistochemistry (Supplemental Fig. 2). The microarray data was analyzed using an R-implementation of rank product analysis [49], defining false discovery rate (FDR) values pertaining to both up- and downregulation for each gene. As expected,

1931

and serving as a control, MXI1 was the top-ranking downregulated gene in both SK-N-BE(2)c and SK-N-SH cells (Table 1). Interestingly, no bona fide MYC targets were found among the most regulated genes. However, as the MYC gene family members are part of a densely regulated network, the effects of MXI1 knock-down might be partly compensated for by other components of this network, and we therefore analyzed the microarray data using gene set enrichment analysis, which would detect small and collective responses on MYC/MYCN targets by MXI1 knock-down (Fig. 6B). For the analyses of MYC-dependent transcription we utilized a gene set pertaining to MYC/MYCN dependent upregulation [52]. Since knock-down of MXI1 should potentiate MYC-driven transcription we expected to find genes annotated as MYC-upregulated to be enriched in the gene lists ranked by upregulation. However, for SK-N-BE(2)c the MYC upregulated gene set was not significantly enriched in the siMXI1 gene list ranked by upregulation (NES = 1.13, FDR q = 0.35) (Fig. 6B). In contrast, analysis of the SK-N-SH data showed that the MYC upregulated gene set was significantly enriched in the si-MXI1 upregulated gene list (NES = 1.44, FDR q = 0.02) (Fig. 6B). In conclusion, these analyses suggest that MXI1 knock-down has a small but significant effect on overall MYC target gene expression, and that the MYCN-amplification status to some degree might influence the MYC-associated role of MXI1 in hypoxic neuroblastoma cells. Due to the very limited changes in expression of classical MYC target genes by MXI1 siRNA detected in the SK-N-BE(2) cells, we further investigated the MYC/MYCN expression and specific activity under hypoxia and the potential role of MXI1 therein. Hypoxia substantially decreased MYCN protein levels in two out of four examined MYCN-amplified neuroblastoma cell lines (Fig. 6C). Expression of MYC in the MYCN-amplified cells was hardly detected, and neither siRNA against MXI1 nor against HIF proteins did significantly alter the MYCN expression (data not shown). The hypoxia-induced downregulation of MYCN protein in SK-N-BE(2)c cells did result in a reduction of total MYC/MYCN-induced transactivation, as determined by the activity of a transfected Luciferase construct containing four MYC/MYCN-binding E-boxes (M4-Luc), after 24 h of hypoxia (Fig. 6D). However, siRNA targeting MXI1 did not affect the hypoxic decrease in E-box activity (Fig. 6D). Expression of the classical MYC/MYCN target gene, ornithine decarboxylase (ODC) [53] was downregulated over time at hypoxia, but this change was unaffected by introduction of siRNA against MXI1 (Fig. 6E). Expression of the reported MYCN target gene MCM7 [54], also decreased slightly in hypoxic SK-N-BE (2)c cells, but again, this pattern was not altered by knocking down MXI1 (Fig. 6E). In MYCN non-amplified SK-N-SH neuroblastoma cells hypoxia did not significantly alter MYC and MYCN expression and accordingly, total E-box activity was largely unaffected by low oxygen or MXI1 siRNA (Fig. 6F). Also, siRNA targeting HIF-1α did not affect the hypoxic E-box reporter activity (Fig. 6F). As seen in MYCNamplified SK-N-BE(2)c cells, a small decrease in ODC expression under hypoxia was displayed in these cells, again without an effect by MXI1 siRNA treatment (Fig. 6G). As neither MAX expression nor levels of the MXI1-related genes, (MAD 1, 3 and 4) demonstrate any specific or consistent changes in hypoxic neuroblastoma cells ([52] and E. F. and S. P., unpublished observations), these results suggest that upregulation of MXI1 is the most pronounced and general hypoxia-induced event within the MYC network and, interestingly,

1932

E XP E RI ME N TA L C E L L R E S EA RC H 315 ( 2 0 0 9 ) 1924 – 193 6

that reducing MXI1 in hypoxic neuroblastoma cells does not overtly affect classical MYC/MYCN target gene expression or specific E-box activity, irrespective of MYCN-amplification status.

Discussion Here we demonstrate direct regulation of the MXI1 gene via HIF transcription factors in hypoxic neuroblastoma cells by identifying the MXI1 HREs to which HIF-1 binds. MXI1 was also rapidly

induced by hypoxia in cultured breast carcinoma cells, indicating a general and important role of MXI1 in adjustment to hypoxic conditions. MXI1 protein was present in both neuroblastoma and DCIS specimens, and high MXI1 immunoreactivity correlated to high HIF-1α levels in neuroblastoma. In summary we show a fast, HIF-dependent transcriptional activation of MXI1, in particular the MXI1-0 isoform. Our data also indicates that acute hypoxia leads to a fast induction of MXI1-0, followed by expression of both the MXI1-0 and MXI1-1 during prolonged hypoxia. Knocking down MXI1 during the acute phase of hypoxia did not substantially affect

E XP E RI ME N TA L C E L L R E S EA RC H 315 ( 2 0 09 ) 19 24 – 193 6

the transcriptional activity of the MYC/MAX network, suggesting that the MXI-0 isoform might have a function separate from the MYCN/MAX antagonizing effect of the MXI-1 isoform. The two major alternatively transcribed isoforms of MXI1 have been identified both in mouse and human [18,19]. Other MXI1 isoforms have also been reported, possibly involving dominantnegative transcripts lacking the DNA-binding exon 3 [55,56]. However, our QPCR analysis of MXI1 exon PCR products did not provide any evidence for additional isoforms in neuroblastoma cells (data not shown). In the present report we detected earlier hypoxic induction of the longer transcript, MXI1-0 (mouse homologue: mxi1-SRα), whereas both isoforms were upregulated at prolonged hypoxia. Specific HIF protein binding was clearly displayed downstream of the first MXI1-0 exon, within an intron separating this exon from the starting MXI1-1 exon, a pattern of HRE localization seen in several known HIF target genes [12]. Interestingly, a recent report [42] suggested HIF-binding within sequences comprised by the MXI1 IVS reporter constructs employed in our analyses (HRE 4). However, our detailed characterization of this genomic region indicated that the HIF-1 binding site reported by Zhang et al., [42] did not significantly contribute to hypoxia-induced MXI1 transcription in neuroblastoma cells. Instead, HREs 2 and 3 detected within the MXI1 IVS were required for full MXI1 induction by HIF-1α in our analyses. These results also highlight the difference between acute and sustained hypoxia. We have previously shown a temporal regulation of HIF-1α and HIF-2α in neuroblastoma cells, where HIF-1α exerts its effect in the acute phase, while HIF-2α is stabilized during prolonged hypoxia and governs much of the hypoxic response over time [14]. In line with these finding, HIF-1α mediates the acute induction of the MXI1-0 promoter, while both HIF-1α and HIF-2α contribute to MXI1-0 expression at later time-points (48 h) (Fig. 2J). Studies of the functional differences between the predominant MXI1-1/mxi1-SRβ and the alternative MXI1-0/mxi1-SRα isoforms show conflicting results. In mouse, mxi1-SRα, with its longer SIN3-interacting domain, has been suggested to be a stronger MYC transcriptional repressor than the shorter mxi1-SRβ counterpart [18]. However, in a recent study it was shown that mxi1-Srα to a large extent lacked the capacity to counteract cellular transformation activity of Myc, which stands in contrast to the potent inhibition exerted by mxi1-SRβ. This functional difference between the two isoforms was also reflected in transient reporter assays, in which only mxi1-SRβ had the capacity to repress the Myc promoter. Our data also gives a plausible

1933

explanation to the observation that the MXI1-0/MXI1-1 ratio was elevated in primary human glioblastomas, since these tumor cells most likely were derived from more or less hypoxic tumor sample [19]. Interestingly, in glioblastoma cells MXI1-0 was shown to be a poor repressor of MYC-dependent transactivation, despite pronounced dimerization with MAX and SIN3 binding [19]. Engstrom et al. [19] indicated cytoplasmic retention as explanation for the inability of over-expressed MXI1-0 to repress MYC transactivation. However, in our stainings for endogenous MXI1 protein we undoubtedly observed strong nuclear MXI1 immunoreactivity in both neuroblastoma and breast carcinoma cells after hypoxic exposure (see Figs. 1 and 5), implicating alternative mechanisms for the limited effects on MYC/MYCN function by MXI1 knock-down. In a study where the basic (DNA-binding) region of MYC was exchanged with the corresponding region of MXI1, results suggested that MYC and MXI1 were not only involved in the regulation of the same genes, but were also controlling distinct sets of genes, indicating MYC-independent MXI1 functions [17,57]. Accordingly, we found specific effects by MXI1 siRNA on several genes previously not reported as direct transcriptional targets of MYC/MYCN (data not shown). This potential MXI1regulated gene expression profile was similar in both analyzed neuroblastoma cell lines irrespective of MYCN-amplification status, which is interesting in light of the recent observation that MYCN and MYC might regulate different subsets of target genes in neuroblastoma cells [58]. Still, independent of MXI1 influence, MYC/MYCN function was found to be reduced by hypoxia, as indicated by ODC downregulation and, in particular, the drop in MYCN levels and E-box reporter activity in hypoxic SK-N-BE(2)c cells. These findings were in agreement with studies showing that hypoxia and HIF-1α protein can counteract MYC function [34,35,42,59,60]. In contrast, HIF-2α, may instead potentiate MYC promoter binding through interaction with MAX, SP1 and MIZ-1 and thereby promote cell cycle progression [34]. We have previously shown that HIF-1α is rapidly stabilized in hypoxic neuroblastoma cells, while HIF-2α is induced upon prolonged hypoxia [14]. Thus, the temporal induction of HIF-1α and HIF-2α during hypoxia might determine the net effect of low oxygen on MYC function and cellular proliferation. Here we also present data showing that the two predominant and functionally distinct MXI1 isoforms, are regulated in a temporal manner in hypoxic neuroblastoma cells. As neither MAX nor any of the MAD genes were regulated at hypoxia, it seems likely that the isoform-specific induction of MXI1 constitute might

Fig. 6 – Knock-down of MXI1 shows limited effects on MYC/MYCN function in hypoxic neuroblastoma cells. (A) QPCR analysis of MXI1-0 expression in SK-N-BE(2)c (top) and SK-N-SH (bottom) cells, transfected with siRNA targeting MXI1 (si-Mx) or control (si-C) and grown under hypoxic or normoxic conditions up to 24 h. (B) Gene set enrichment analysis of known MYC target genes in microarray data from MXI1 knock-down SK-N-BE(2)c and SK-N-SH cells. Genes in the MXI1 siRNA data lists were ranked, for upregulation, by measure of rank product analysis FDR. In SK-N-BE(2)c cells (upper panels) no significant enrichment was found for the MYC target genes (NES = 1.13, FDR q = 0.35). For SK-N-SH cells the MYC gene set was significantly enriched (NES = 1.44, FDR q = 0.02). (C) Western blot of four MYCN-amplified cell lines, SK-N-BE(2)c, KCN-69n, IMR-32 and LA-N-5, cultured at 1% or 21% oxygen up to 48 h and analyzed for MYCN protein content. (D) SK-N-BE(2)c cells were transfected with a vector containing 4 consecutive E-boxes, coupled to a Luciferase reporter (M4-Luc), together with siRNAs against MXI1 or control. Cells were grown at 1 or 21% O2 for 24 h before determination of Luciferase activities, which were normalized for each individual treatment (hypoxia/ normoxia). Results are representative of two independent experiments and error bars indicate ± SD within triplicate transfections. (E) QPCR analysis of ODC and MCM7 expression in normoxic and hypoxic SK-N-BE(2)c cells transfected with mock, control or MXI1 siRNA. (F) Similar M4-Luc E-box reporter gene assay as in (D), here performed in hypoxic and normoxic (24 h) SK-N-SH cells transfected with siRNAs targeting control, MXI1 or HIF-1α. Error bars show ± SD within triplicate transfections. (G) ODC expression, investigated by QPCR in MXI1 siRNA-transfected SK-N-SH cells, cultured at 1 or 21% oxygen.

1934

E XP E RI ME N TA L C E L L R E S EA RC H 315 ( 2 0 0 9 ) 1924 – 193 6

Table 1 – Genes identified as either up- or downregulated by a combined rank product analysis (FDR = 0). Downregulated genes

Mean downregulation

Upregulated genes

Mean upregulation

Rank

Gene symbol

Gene ID

SK-N-BE(2)c

SK-N-SH

Rank

Gene symbol

Gene ID

SK-N-BE(2)c

SK-N-SH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

MXI1 AK3L1 DNAL1 TSSC1 C1orf43 GOLPH4 GNG10 IGFBP5 HRAS FAM63B IGFBP5 RAB40B TTC12 FLJ25967 TMEM81 HSF4 C16orf28 AK3L1 NKX2-5 NTNG2 DCTN4 MIB1 UHMK1 LPGAT1 LIFR RP3-402G11.5 NUDT14 LSAMP ADARB1 DYNC1LI2 TSPAN13 SLC4A3 SYT5 GM2A DYNC1LI2 IRF6 CRYBA2 MALAT1 SLIT1 CHRM1 MPP5 TPCN1 SMPD3 DEDD BTRC CX3CL1 WDR18 ADPGK

4601 205 83544 7260 25912 27333 2790 3488 3265 54629 3488 10966 54970 440823 388730 3299 65259 205 1482 84628 51164 57534 127933 9926 3977 83642 256281 4045 104 1783 27075 6508 6861 2760 1783 3664 1412 378938 6585 1128 64398 53373 55512 9191 8945 6376 57418 83440

−2.1 −1.9 −2.1 −1.7 −1.5 −1.7 −1.5 −1.2 −1.3 −1.9 −1.1 −0.8 −1.1 −0.9 −1.5 −1.1 −1.3 −1.3 −1.0 −0.6 −1.3 −0.8 −1.0 −1.4 −1.0 −1.4 −0.9 −0.6 −1.1 −1.0 −1.0 −0.9 −0.6 −1.5 −0.9 −1.1 −1.0 −0.9 −1.2 −1.4 −0.9 −1.0 −1.2 −1.0 −0.9 −1.5 −1.3 −1.1

−1.5 −1.3 −1.1 −1.0 −1.1 −1.0 −1.1 −1.3 −1.1 −0.8 −1.2 −1.4 −1.1 −1.2 −0.8 −1.0 −0.9 −0.8 −1.1 −1.4 −0.8 −1.1 −0.9 −0.7 −0.9 −0.7 −1.0 −1.2 −0.8 −0.9 −0.9 −0.9 −1.1 −0.7 −0.9 −0.7 −0.8 −0.8 −0.6 −0.5 −0.8 −0.8 −0.6 −0.7 −0.8 −0.4 −0.5 −0.7

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

LUM TAX1BP3 GALNT1 DKK1 SLC7A11 H2AFV BAALC ARHGAP10 CCDC25 MND1 BNIP2 UQCRH LGR5 ADAMTS3 ADAM12 IFRD2 H2AFV LITAF SSTR2 HMGB3 HMGB3 E2F7 DKFZp667G211 CEP55 SCGN CUTC KCNH2 MT1E CSNK2A1 RBPMS PLEKHA2 VGLL2 SHANK2 DPYSL3

4060 30851 2589 22943 23657 94239 79870 79658 55246 84057 663 7388 8549 9508 8038 7866 94239 9516 6752 3149 3149 144455 131544 55165 10590 51076 3757 4493 1457 11030 59339 245806 22941 1809

1.0 1.1 1.2 1.3 1.5 0.9 0.4 1.9 1.1 1.1 1.0 1.1 0.9 0.8 0.4 0.7 0.8 1.0 1.0 0.9 0.9 0.6 0.5 0.6 −0.2 0.7 0.6 0.2 0.6 0.6 0.6 1.1 0.8 0.5

2.0 1.0 0.9 0.8 0.5 0.9 1.9 0.6 0.8 0.7 0.7 0.7 0.8 1.0 1.3 0.9 0.8 0.6 0.6 0.6 0.6 0.9 1.1 0.9 1.9 0.9 0.9 1.6 1.0 1.0 0.9 0.5 0.7 0.9

SK-N-BE(2)c and SK-N-SH neuroblastoma cells were transfected with siRNA against either MXI1 or control, and grown at 1% O2 for 24 h. RNA was hybridized to oligo microarrays representing app. 26,000 genes and ESTs. Rank refers to rank product analysis score and Gene ID refers to the NCBI Entrez Gene database. Mean up- and downregulation is given in log(2) values of three independent replicates.

represent an important function in hypoxic adaptation that might fine-tune the activity of the MYC/MAX/MAD network. However, it also remains possible that MXI1-0 may exert effects independent of this network. In a recent study it was shown that MXI1 was elevated in renal cell carcinoma, a tumor type that due to loss of the VHL gene is characterized normoxic stabilization of HIF proteins and hence a constitutive hypoxic response. However, ablation of MXI1 in this cellular context

clearly perturbed growth, both in vitro and in vivo. Whether these observations in renal cell carcinomas are related to Mycdependent or -independent functions remains to be clarified [57]. These results nevertheless corroborate our present findings in neuroblastoma cells and indicate that MXI1 represents an important HIF target gene that provides a new level of complexity in the sophisticated interplay between the HIF and MYC functions in hypoxic cells.

E XP E RI ME N TA L C E L L R E S EA RC H 315 ( 2 0 09 ) 19 24 – 193 6

Acknowledgments This work was supported by the Swedish Cancer Society, the Children's Cancer Foundation of Sweden, the Swedish Research Council, the Swedish Foundation for Strategic Research, the Research Program in Medical Bioinformatics of the Swedish Knowledge Foundation, Ollie and Elof Ericsson's, Crafoord's, Hans von Kantzow's and Gunnar Nilsson's foundations, the research funds of Malmö University Hospital and grants RD06/0020/0102 and PI06/ 1576 from Instituto Carlos III, Madrid, Spain.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.yexcr.2009.02.015.

REFERENCES

[1] A.L. Harris, Hypoxia—a key regulatory factor in tumour growth, Nat. Rev. Cancer 2 (2002) 38–47. [2] G.L. Semenza, Targeting HIF-1 for cancer therapy, Nat. Rev. Cancer 3 (2003) 721–732. [3] R.K. Bruick, S.L. McKnight, A conserved family of prolyl-4-hydroxylases that modify HIF, Science 294 (2001) 1337–1340. [4] A.C. Epstein, J.M. Gleadle, L.A. McNeill, K.S. Hewitson, J. O'Rourke, D.R. Mole, M. Mukherji, E. Metzen, M.I. Wilson, A. Dhanda, Y.M. Tian, N. Masson, D.L. Hamilton, P. Jaakkola, R. Barstead, J. Hodgkin, P.H. Maxwell, C.W. Pugh, C.J. Schofield, P.J. Ratcliffe, C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation, Cell 107 (2001) 43–54. [5] M. Ivan, K. Kondo, H. Yang, W. Kim, J. Valiando, M. Ohh, A. Salic, J.M. Asara, W.S. Lane, W.G. Kaelin Jr., HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing, Science 292 (2001) 464–468. [6] P. Jaakkola, D.R. Mole, Y.M. Tian, M.I. Wilson, J. Gielbert, S.J. Gaskell, A. Kriegsheim, H.F. Hebestreit, M. Mukherji, C.J. Schofield, P.H. Maxwell, C.W. Pugh, P.J. Ratcliffe, Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation, Science 292 (2001) 468–472. [7] L.E. Huang, J. Gu, M. Schau, H.F. Bunn, Regulation of hypoxia-inducible factor 1alpha is mediated by an O2-dependent degradation domain via the ubiquitin–proteasome pathway, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 7987–7992. [8] P.J. Kallio, W.J. Wilson, S. O'Brien, Y. Makino, L. Poellinger, Regulation of the hypoxia-inducible transcription factor 1alpha by the ubiquitin–proteasome pathway, J. Biol. Chem. 274 (1999) 6519–6525. [9] P.H. Maxwell, M.S. Wiesener, G.W. Chang, S.C. Clifford, E.C. Vaux, M.E. Cockman, C.C. Wykoff, C.W. Pugh, E.R. Maher, P.J. Ratcliffe, The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis, Nature 399 (1999) 271–275. [10] D. Lando, D.J. Peet, J.J. Gorman, D.A. Whelan, M.L. Whitelaw, R.K. Bruick, FIH-1 is an asparaginyl hydroxylase enzyme that regulates the transcriptional activity of hypoxia-inducible factor, Genes Dev. 16 (2002) 1466–1471. [11] P.C. Mahon, K. Hirota, G.L. Semenza, FIH-1: a novel protein that interacts with HIF-1alpha and VHL to mediate repression of HIF-1 transcriptional activity, Genes Dev. 15 (2001) 2675–2686. [12] R.H. Wenger, D.P. Stiehl, G. Camenisch, Integration of oxygen signaling at the consensus HRE, Sci. STKE 2005 (2005) re12.

1935

[13] E. Fredlund, M. Ovenberger, K. Borg, S. Pahlman, Transcriptional adaptation of neuroblastoma cells to hypoxia, Biochem. Biophys. Res. Commun. 366 (2008) 1054–1060. [14] L. Holmquist-Mengelbier, E. Fredlund, T. Lofstedt, R. Noguera, S. Navarro, H. Nilsson, A. Pietras, J. Vallon-Christersson, A. Borg, K. Gradin, L. Poellinger, S. Pahlman, Recruitment of HIF-1alpha and HIF-2alpha to common target genes is differentially regulated in neuroblastoma: HIF-2alpha promotes an aggressive phenotype, Cancer Cell. 10 (2006) 413–423. [15] N. Schreiber-Agus, L. Chin, K. Chen, R. Torres, G. Rao, P. Guida, A.I. Skoultchi, R.A. DePinho, An amino-terminal domain of Mxi1 mediates anti-Myc oncogenic activity and interacts with a homolog of the yeast transcriptional repressor SIN3, Cell 80 (1995) 777–786. [16] A.S. Zervos, J. Gyuris, R. Brent, Mxi1, a protein that specifically interacts with Max to bind Myc–Max recognition sites, Cell 72 (1993) 223–232. [17] R.C. O'Hagan, N. Schreiber-Agus, K. Chen, G. David, J.A. Engelman, R. Schwab, L. Alland, C. Thomson, D.R. Ronning, J.C. Sacchettini, P. Meltzer, R.A. DePinho, Gene-target recognition among members of the myc superfamily and implications for oncogenesis, Nat. Genet. 24 (2000) 113–119. [18] C. Dugast-Darzacq, M. Pirity, J.K. Blanck, A. Scherl, N. Schreiber-Agus, Mxi1-SRalpha: a novel Mxi1 isoform with enhanced transcriptional repression potential, Oncogene 23 (2004) 8887–8899. [19] L.D. Engstrom, A.S. Youkilis, J.L. Gorelick, D. Zheng, V. Ackley, C.A. Petroff, L.Q. Benson, M.R. Coon, X. Zhu, S.M. Hanash, D.S. Wechsler, Mxi1-0, an alternatively transcribed Mxi1 isoform, is overexpressed in glioblastomas, Neoplasia 6 (2004) 660–673. [20] C. Dugast-Darzacq, T. Grange, N.B. Schreiber-Agus, Differential effects of Mxi1-SRalpha and Mxi1-SRbeta in Myc antagonism, FEBS J. 274 (2007) 4643–4653. [21] N. Schreiber-Agus, Y. Meng, T. Hoang, H. Hou Jr., K. Chen, R. Greenberg, C. Cordon-Cardo, H.W. Lee, R.A. DePinho, Role of Mxi1 in ageing organ systems and the regulation of normal and neoplastic growth, Nature 393 (1998) 483–487. [22] M.M. Taj, R.J. Tawil, L.D. Engstrom, Z. Zeng, C. Hwang, M.G. Sanda, D.S. Wechsler, Mxi1, a Myc antagonist, suppresses proliferation of DU145 human prostate cells, Prostate 47 (2001) 194–204. [23] D.S. Wechsler, C.A. Shelly, C.A. Petroff, C.V. Dang, MXI1, a putative tumor suppressor gene, suppresses growth of human glioblastoma cells, Cancer Res. 57 (1997) 4905–4912. [24] S. Edelhoff, D.E. Ayer, A.S. Zervos, E. Steingrimsson, N.A. Jenkins, N.G. Copeland, R.N. Eisenman, R. Brent, C.M. Disteche, Mapping of two genes encoding members of a distinct subfamily of MAX interacting proteins: MAD to human chromosome 2 and mouse chromosome 6, and MXI1 to human chromosome 10 and mouse chromosome 19, Oncogene 9 (1994) 665–668. [25] E.V. Prochownik, L. Eagle Grove, D. Deubler, X.L. Zhu, R.A. Stephenson, L.R. Rohr, X. Yin, A.R. Brothman, Commonly occurring loss and mutation of the MXI1 gene in prostate cancer, Genes Chromosomes Cancer 22 (1998) 295–304. [26] B.K. Rasheed, R.E. McLendon, H.S. Friedman, A.H. Friedman, H.E. Fuchs, D.D. Bigner, S.H. Bigner, Chromosome 10 deletion mapping in human gliomas: a common deletion region in 10q25, Oncogene 10 (1995) 2243–2246. [27] D.K. Scott, D. Straughton, M. Cole, S. Bailey, D.W. Ellison, S.C. Clifford, Identification and analysis of tumor suppressor loci at chromosome 10q23.3–10q25.3 in medulloblastoma, Cell Cycle 5 (2006) 2381–2389. [28] K.G. Hermans, D.C. van Alewijk, J.A. Veltman, W. van Weerden, A.G. van Kessel, J. Trapman, Loss of a small region around the PTEN locus is a major chromosome 10 alteration in prostate cancer xenografts and cell lines, Genes Chromosomes Cancer 39 (2004) 171–184. [29] L.R. Eagle, X. Yin, A.R. Brothman, B.J. Williams, N.B. Atkin, E.V. Prochownik, Mutation of the MXI1 gene in prostate cancer, Nat. Genet. 9 (1995) 249–255.

1936

E XP E RI ME N TA L C E L L R E S EA RC H 315 ( 2 0 0 9 ) 1924 – 193 6

[30] D. Bartsch, S.L. Peiffer, Z. Kaleem, S.A. Wells Jr., P.J. Goodfellow, Mxi1 tumor suppressor gene is not mutated in primary pancreatic adenocarcinoma, Cancer Lett. 102 (1996) 73–76. [31] S.M. Edwards, D.P. Dearnaley, A. Ardern-Jones, R.A. Hamoudi, D.F. Easton, D. Ford, R. Shearer, A. Dowe, R.A. Eeles, No germline mutations in the dimerization domain of MXI1 in prostate cancer clusters. The CRC/BPG UK Familial Prostate Cancer Study Collaborators. Cancer Research Campaign/British Prostate Group, Br. J. Cancer 76 (1997) 992–1000. [32] N. Kawamata, D. Park, S. Wilczynski, J. Yokota, H.P. Koeffler, Point mutations of the Mxil gene are rare in prostate cancers, Prostate 29 (1996) 191–193. [33] X.J. Li, D.Y. Wang, Y. Zhu, R.J. Guo, X.D. Wang, K. Lubomir, K. Mukai, H. Sasaki, H. Yoshida, T. Oka, R. Machinami, K. Shinmura, M. Tanaka, H. Sugimura, Mxi1 mutations in human neurofibrosarcomas, Jpn. J. Cancer Res. 90 (1999) 740–746. [34] J.D. Gordan, J.A. Bertout, C.J. Hu, J.A. Diehl, M.C. Simon, HIF-2alpha promotes hypoxic cell proliferation by enhancing c-myc transcriptional activity, Cancer Cell 11 (2007) 335–347. [35] M. Koshiji, Y. Kageyama, E.A. Pete, I. Horikawa, J.C. Barrett, L.E. Huang, HIF-1alpha induces cell cycle arrest by functionally counteracting Myc, EMBO J. 23 (2004) 1949–1956. [36] G.M. Brodeur, Neuroblastoma: biological insights into a clinical enigma, Nat. Rev. Cancer 3 (2003) 203–216. [37] L. Chen, T. Fink, P. Ebbesen, V. Zachar, Temporal transcriptome of mouse ATDC5 chondroprogenitors differentiating under hypoxic conditions, Exp. Cell Res. 312 (2006) 1727–1744. [38] P.G. Corn, M.S. Ricci, K.A. Scata, A.M. Arsham, M.C. Simon, D.T. Dicker, W.S. El-Deiry, Mxi1 is induced by hypoxia in a HIF-1-dependent manner and protects cells from c-Myc-induced apoptosis, Cancer Biol. Ther. 4 (2005) 1285–1294. [39] N.C. Denko, L.A. Fontana, K.M. Hudson, P.D. Sutphin, S. Raychaudhuri, R. Altman, A.J. Giaccia, Investigating hypoxic tumor physiology through gene expression patterns, Oncogene 22 (2003) 5907–5914. [40] E.N. Maina, M.R. Morris, M. Zatyka, R.R. Raval, R.E. Banks, F.M. Richards, C.M. Johnson, E.R. Maher, Identification of novel VHL target genes and relationship to hypoxic response pathways, Oncogene 24 (2005) 4549–4558. [41] V. Wang, D.A. Davis, M. Haque, L.E. Huang, R. Yarchoan, Differential gene up-regulation by hypoxia-inducible factor-1alpha and hypoxia-inducible factor-2alpha in HEK293T cells, Cancer Res. 65 (2005) 3299–3306. [42] H. Zhang, P. Gao, R. Fukuda, G. Kumar, B. Krishnamachary, K.I. Zeller, C.V. Dang, G.L. Semenza, HIF-1 inhibits mitochondrial biogenesis and cellular respiration in VHL-deficient renal cell carcinoma by repression of C-MYC activity, Cancer Cell 11 (2007) 407–420. [43] K. De Preter, F. Speleman, V. Combaret, J. Lunec, G. Laureys, B.H. Eussen, N. Francotte, J. Board, A.D. Pearson, A. De Paepe, N. Van Roy, J. Vandesompele, Quantification of MYCN, DDX1, and NAG gene copy number in neuroblastoma using a real-time quantitative PCR assay, Mod. Pathol. 15 (2002) 159–166. [44] K. Helczynska, A. Kronblad, A. Jogi, E. Nilsson, S. Beckman, G. Landberg, S. Pahlman, Hypoxia promotes a dedifferentiated phenotype in ductal breast carcinoma in situ, Cancer Res. 63 (2003) 1441–1444. [45] T. Lofstedt, A. Jogi, M. Sigvardsson, K. Gradin, L. Poellinger, S. Pahlman, H. Axelson, Induction of ID2 expression by hypoxia-inducible factor-1: a role in dedifferentiation of hypoxic neuroblastoma cells, J. Biol. Chem. 279 (2004) 39223–39231.

[46] G. Jonsson, J. Staaf, E. Olsson, M. Heidenblad, J. Vallon-Christersson, K. Osoegawa, P. de Jong, S. Oredsson, M. Ringner, M. Hoglund, A. Borg, High-resolution genomic profiles of breast cancer cell lines assessed by tiling BAC array comparative genomic hybridization, Genes Chromosomes Cancer 46 (2007) 543–558. [47] L.H. Saal, C. Troein, J. Vallon-Christersson, S. Gruvberger, A. Borg, C. Peterson, BioArray Software Environment (BASE): a platform for comprehensive management and analysis of microarray data, Genome Biol. 3 (2002) SOFTWARE0003. [48] Y.H. Yang, S. Dudoit, P. Luu, D.M. Lin, V. Peng, J. Ngai, T.P. Speed, Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation, Nucleic Acids Res. 30 (2002) e15. [49] R. Breitling, P. Armengaud, A. Amtmann, P. Herzyk, Rank products: a simple, yet powerful, new method to detect differentially regulated genes in replicated microarray experiments, FEBS Lett. 573 (2004) 83–92. [50] A. Subramanian, P. Tamayo, V.K. Mootha, S. Mukherjee, B.L. Ebert, M.A. Gillette, A. Paulovich, S.L. Pomeroy, T.R. Golub, E.S. Lander, J.P. Mesirov, Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 15545–15550. [51] K.I. Zeller, A.G. Jegga, B.J. Aronow, K.A. O'Donnell, C.V. Dang, An integrated database of genes responsive to the Myc oncogenic transcription factor: identification of direct genomic targets, Genome Biol. 4 (2003) R69. [52] E. Fredlund, M. Ringner, J.M. Maris, S. Pahlman, High Myc pathway activity and low stage of neuronal differentiation associate with poor outcome in neuroblastoma, Proc. Natl. Acad. Sci. U. S. A. 105 (2008) 14094–14099. [53] C. Bello-Fernandez, G. Packham, J.L. Cleveland, The ornithine decarboxylase gene is a transcriptional target of c-Myc, Proc. Natl. Acad. Sci. U. S. A. 90 (1993) 7804–7808. [54] J.M. Shohet, M.J. Hicks, S.E. Plon, S.M. Burlingame, S. Stuart, S.Y. Chen, M.K. Brenner, J.G. Nuchtern, Minichromosome maintenance protein MCM7 is a direct target of the MYCN transcription factor in neuroblastoma, Cancer Res. 62 (2002) 1123–1128. [55] N. Kawamata, K.J. Sugimoto, S. Sakajiri, K. Oshimi, H.P. Koeffler, Mxi1 isoforms are expressed in hematological cell lines and normal bone marrow, Int. J. Oncol. 26 (2005) 1369–1375. [56] D.S. Wechsler, C.A. Shelly, C.V. Dang, Genomic organization of human MXI1, a putative tumor suppressor gene, Genomics 32 (1996) 466–470. [57] C.C. Tsao, B.T. Teh, E. Jonasch, N. Schreiber-Agus, E. Efstathiou, A. Hoang, B. Czerniak, C. Logothetis, P.G. Corn, Inhibition of Mxi1 suppresses HIF-2alpha-dependent renal cancer tumorigenesis, Cancer Biol. Ther. 7 (2008) 1619–1627. [58] F. Westermann, D. Muth, A. Benner, T. Bauer, K.O. Henrich, A. Oberthuer, B. Brors, T. Beissbarth, J. Vandesompele, F. Pattyn, B. Hero, R. Konig, M. Fischer, M. Schwab, Distinct transcriptional MYCN/c-MYC activities are associated with spontaneous regression or malignant progression in neuroblastomas, Genome Biol. 9 (2008) R150. [59] A. Kaidi, A.C. Williams, C. Paraskeva, Interaction between beta-catenin and HIF-1 promotes cellular adaptation to hypoxia, Nat. Cell Biol. 9 (2007) 210–217. [60] F.A. Mack, J.H. Patel, M.P. Biju, V.H. Haase, M.C. Simon, Decreased growth of Vhl−/− fibrosarcomas is associated with elevated levels of cyclin kinase inhibitors p21 and p27, Mol. Cell Biol. 25 (2005) 4565–4578.