Aberrant RSPO3-LGR4 Signaling in Keap1-Deficient Lung

ORIGINAL ARTICLE Aberrant RSPO3-LGR4 signaling in Keap1-deﬁcient lung adenocarcinomas promotes tumor aggressiveness

X Gong1,2,JYi1,2, KS Carmon1,2, CA Crumbley1,2, W Xiong1,2, A Thomas1,2,XFan1,2, S Guo3,ZAn1,2, JT Chang1,4,5,6 and QJ Liu1,2

The four R-spondins (RSPO1–4) and their three related receptors LGR4, 5 and 6 (LGR4–6) have emerged as a major ligand-receptor system with critical roles in development and stem cell survival through modulation of Wnt signaling. Recurrent, gain-of-expression gene fusions of RSPO2 (to EIF3E) and RSPO3 (to PTPRK) occur in a subset of human colorectal cancer. However, the exact roles and mechanisms of the RSPO-LGR system in oncogenesis remain largely unknown. We found that RSPO3 is aberrantly expressed at high levels in approximately half of Keap1-mutated lung adenocarcinomas (ADs). This high RSPO3 expression is driven by a combination of demethylation of its own promoter region and deficiency in Keap1 instead of gene fusion as in colon cancer. Patients with RSPO3-high tumors (~9%, 36/412) displayed much poorer survival than the rest of the cohort (median survival of 28 vs 163 months, log-rank test Po0.0001). Knockdown (KD) of RSPO3, LGR4 or their signaling mediator IQGAP1 in lung cancer cell lines with Keap1 deficiency and high RSPO3-LGR4 expression led to reduction in cell proliferation and migration in vitro, and KD of LGR4 or IQGAP1 resulted in decrease in tumor growth and metastasis in vivo. These findings suggest that aberrant RSPO3-LGR4 signaling potentially acts as a driving mechanism in the aggressiveness of Keap1-deficient lung ADs.

Oncogene (2015) 34, 4692–4701; doi:10.1038/onc.2014.417; published online 22 December 2014

INTRODUCTION the formation of a supercomplex between RSPO-LGR4 and Wnt R-spondins are a group of four highly related secreted proteins receptors. In this configuration, IQGAP1 brings in MEK1/2 to (RSPO1–4) with critical roles in embryonic development phosphorylate LRP5/6 for the β-catenin-dependent pathway and and organogenesis as well as in the self-renewal and survival of N-WASP/mDia1 to coordinate actin dynamics for the β-catenin- adult stem cells.1 In particular, loss of RSPO2 led to hypoplasia independent pathway.11 and reduced branching of the lung during mouse Dysregulation of Wnt signaling occurs in nearly every major development.2,3 Work from us and others demonstrated that type of solid tumors. Gain-of-expression gene fusions of RSPO2 (to RSPOs activate three related receptors LGR4–6 (leucine-rich EIF3E) and RSPO3 (to PTPRK) were identified in 10% (7/68) of repeat-containing, G protein-coupled receptor 4, 5 and 6) to human colon cancer.14 The fusions were inferred to have a driving 4–6 potentiate Wnt signaling. LGR4–6 contain a large extracellular role in the carcinogenesis of the affected tumors because of their domain with 17 leucine-rich repeats and a seven transmembrane recurrent occurrence and exclusivity with Apc/β-catenin mutat- domain homologous to members of the rhodopsin family of G 14 7–9 ions. In MMTV-induced mouse models of breast and colon cancer, protein-coupled receptors. LGR4-bound RSPOs directly interact RSPO2 and RSPO3 were two of the most frequent viral integration with two membrane-bound E3 ligases (RNF43 and ZNRF3), which 10 sites, and ectopic expression of RSPO2/3 in mouse mammary otherwise ubiquitinate Fzd receptors for degradation. Formation epithelial cells increased tumor formation and metastasis.15–17 of the LGR4-RSPO-RNF43/ZNRF3 ternary complex induces Furthermore, knockout of LGR4 in mice led to profound hypoplasia the clearance of the E3 ligases, leading to reduced ubiquitination and impaired tubulogenesis in multiple organs during develop- and eventually elevated levels of Wnt receptors on the cell 18–20 surface and increased Wnt signaling.10 Just recently, we ment, suggesting a critical role of LGR4 in the regulation of cell identified IQGAP1 as an LGR4-binding protein and showed that proliferation and migration. Intriguingly, LGR4 was found to be it plays an essential role in RSPO-LGR4-induced potentiation highly upregulated in both adenocarcinomas (AD) and squamous of Wnt signaling.11 IQGAP1 is an intracellular scaffold protein cell carcinomas of non-small-cell lung cancer (NSCLC) despite low 21 that binds to and modulates the activities of a plethora of expression in normal adult lung. We found that RSPO3 was highly signaling molecules to regulate cell adhesion and migration.12,13 expressed in a subset of ADs. Here we show that the aberrant RSPO3 We found that RSPO-LGR4 not only induces the clearance of expression in lung ADs was not driven by PTPRK fusion as in colon RNF43/ZNRF3 but also increases the affinity of IQGAP1 for cancer, and that RSPO3-LGR4 signaling plays a major role in the dishelvelled (DVL) bound to the Wnt signalosome. This leads to aggressiveness of RSPO3-high tumors.

1Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center at Houston, Houston, TX, USA; 2Texas Therapeutics Institute, University of Texas Health Science Center at Houston, Houston, TX, USA; 3Divison of Oncology, University of Texas Health Science Center at Houston, Houston, TX, USA; 4Center for Clinical and Translational Sciences, University of Texas Health Science Center at Houston, Houston, TX, USA; 5Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Houston, TX, USA and 6School of Biomedical Informatics, University of Texas Health Science Center at Houston, Houston, TX, USA. Correspondence: Dr QJ Liu, Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center at Houston, 1825 Pressler Street, Suite 530A, Houston, TX 77030, USA. E-mail: [email protected] Received 29 April 2014; revised 31 October 2014; accepted 3 November 2014; published online 22 December 2014 RSPO3-LGR4 signaling in lung cancer X Gong et al 4693 RESULTS (CCLE) project24 (Supplementary Figure 1b). Using our recently 25 RSPO3 is aberrantly expressed in a subset of lung ADs and its high characterized LGR4 monoclonal antibody 7E7, we analyzed the expression is associated with poor survival expression of LGR4 in normal and AD samples of the lung on We mined the RNA-Seq data of LGR4–6, RSPO1–4, and other tissue microarrays. LGR4 staining was detected in all AD samples genes encoding Wnt ligands, receptors and modulators in TCGA’s with two representatives shown (Figures 1a and b). No expression of LGR4 was observed in respiratory epithelial or alveolar cells in provisional Lung AD cohort (LUAD, 230 samples) as provided by 22 normal adult lung (Figure 1c), consistent with previous report of the cBio Portal website. Based on the RSEM values (RNA-Seq by 21 23 LGR4 upregulation in lung ADs. Expectation-Maximization) of the transcripts, LGR4 was Of the potential RSPO ligands, RSPO3 was expressed at expressed at moderate to high levels in ~ 90% of the cases, signiﬁcant levels with a highly skewed pattern, that is, high levels whereas LGR5 and LGR6 were expressed at much lower levels in in ~ 10% (25/230) of the tumors with no to little expression in the nearly all samples (Supplementary Figure 1a). A similar pattern of rest of the cases (Figure 1d). The other three RSPOs were expression for LGR4–6 was also found in the 186 lung cancer cell expressed at much lower, insigniﬁcant levels with two exceptions line collection characterized by the Cancer Cell Line Encyclopedia in RSPO2 (Supplementary Figure 1c). Comparison of the RSPO3

Tumor-1 Tumor-2 Normal lung

15 15 RSPO3-CCLE 100 RSPO3-high (One-way ANOVA p = 0.2) RSPO3-TCGA RSPO3-norm 12 LGR4-TCGA 80 (Logrank test p = 0.003) 10 60 9

40 % Surviving 5

Exp (log2[signal]) 6 20 N = 173 RSPO3 Exp (log2[RSEM]) N = 22 3 0 0 0 50 100 150 200 250 0 50 100 150 200 250 Sample # Months of Survival Stage-I Stge-II Stage-III Stage-IV

120 120 RSPO3-high RSPO3-high 100 RSPO3-high 100 100 RSPO3-norm RSPO3-norm RSPO3-norm 80 (Logrank test p < 0.0001) 80 (Logrank test p = 0.01) 80 (Logrank test p = 0.01) 60 60 60

40 % Surviving % Surviving 40 % Surviving 40 N = 376

N = 44 20 20 N = 127 20 N = 36 N = 14 N = 5 0 0 0 0 50 100 150 200 250 0 50 100 150 200 250 0 50 100 150 200 250 Months of Survival Months of Survival Months of Survival Figure 1. High level of RSPO3 expression is correlated with poor survival in lung AD. (a-c) Immunohistochemistry staining of LGR4 in two lung AD samples (a, b) and normal adult lung (c). (d) Expression distribution of RSPO3 and LGR4 in 230 lung AD samples of TCGA’s LUAD cohort and RSPO3 in CCLE’s 186 lung cancer cell lines. Values in log2 of expression signal (RSEM for TCGA samples and RMA for CCLE samples) were sorted from low to high for each gene and plotted. (e) Kaplan–Meier survival plot of patients with tumors of high RSPO3 expression vs the rest of the cohort in the TCGA LUAD set. RSPO3-high was deﬁned as samples with log2[RSEM] values at 1 s.d. above the population mean (Z 4= +1) while the remaining samples were deﬁned as RSPO-norm. (f) Scatter plot of RSPO3 expression among different tumor stages in TCGA’s LUAD cohort. (g) Kaplan–Meier survival plot of patients with RSPO3-high vs RSPO3-norm tumors in stages I-II of TCGA’s LUAD cohort. (h) Kaplan–Meier survival plot of patients with RSPO3-high vs RSPO3-norm tumors in stages III-IV of TCGA’s LUAD cohort. (i) Kaplan–Meier survival plot of data pooled from three lung cancer studies (TCGA LUAD, GSE3141 and GSE31210).

© 2015 Macmillan Publishers Limited Oncogene (2015) 4692 – 4701 RSPO3-LGR4 signaling in lung cancer X Gong et al 4694 expression data between tumors and their matched normal correlation between upregulation of RSPO3 expression (defined tissues revealed that the high RSPO3 expression in tumors was those with Z4+1 by cBioPortal) and deficiency in Keap1 (Fisher’s due to upregulation (Supplementary Figure 2). Furthermore, the exact test P = 0; Supplementary Figure 5a). No association was distributions of RSPO1–4 in CCLE’s lung cancer cell line collection found between high expression of RSPO3 and the mutation status are also similar to those in primary tumors (Figure 1d and of other cancer genes, such as TP53, KRAS and EGFR Supplementary Figure 1d), that is, RSPO3 is highly expressed in a (Supplementary Figure 5a). Keap1 is a negative regulator of the subset (17%, 32/186) of the cell lines using a cutoff value of 4 × transcription factor NRF2 (encoded by NFE2L2) and is mutated in – above background, whereas the other three RSPOs are rarely NSCLC with an overall frequency of ~ 15%.29 31 Keap1 and NRF2 expressed. Taken together, these data indicate that LGR4 is abnormalities were previously shown to be associated with poor abundantly expressed in the majority of primary lung ADs with a outcome in NSCLC patients.32,33 In this TCGA LUAD cohort, Keap1 subset of them co-expressing high levels of RSPO3. was mutated or downregulated in 17% (38/230, mutated = 24, low RSPO-LGR4-IQGAP1 signaling has potent effects on cell expression is estimated to be 14) of the tumors, whereas no proliferation, adhesion and migration.11 As metastasis is the major mutation or upregulation of NFE2L2 was seen. Remarkably, 20 of cause of cancer-related death, we asked if high RSPO3 expression the 25 RSPO3-high tumors had loss of Keap1 function because of is associated with patient survival. In univariate analysis of the either mutation or low expression (Figure 2a). The remaining five TCGA LUAD cohort, patients with RSPO3-high tumors (defined as RSPO3-high tumors had no sequencing data on Keap1 at the time those with log2-RSEM values of RSPO3 at 1 standard deviation of this analysis, but all had high expression of Keap1/NRF2- above the mean, that is, Z = +1) showed much worse overall regualted genes (Figure 2a), suggesting that gain of NRF2 activity survival than the rest of the cohort (Figure 1e, median survival of occurred in these tumors. We also carried out a global analysis of 22 vs 49 months, log-rank test P = 0.008; hazard ratio (HR) = 3.8, genes that were associated with high RSPO3 expression in the 95% confidence interval (CI) = 1.7–8.8). In multivariate analysis TCGA LUAD set. The most prevalent finding is that RSPO3-high using a continuous variable Cox proportional hazards model, tumors were highly enriched in genes regulated by the Keap1- RSPO3 was found to be predictive of outcomes independent of NRF2 pathway, such as ABCC2, AKR1C1 and AKR1C2 (Figure 2a). gender, age and smoking status (P = 0.03, HR = 2.1, 95% CI = 1.1– These data unequivocally show that high RSPO3 expression only 4.3). The incidence of RSPO3-high tumors did not vary significantly occurred in a subset (approximately half) of Keap1-deficient among tumor stages (Figure 1f) and high RSPO3 expression was tumors. We then asked if RSPO3 expression level differentiated significantly correlated with poor survival in both early (I-II) and survival of patients with Keap1-deficient tumors. Patients with late (III-IV) stages of the cancer (Figures 1g and h), suggesting that tumors containing both high RSPO3 expression and Keap1 upregulation of RSPO3 occurred as an early event during tumor deficiency showed significantly shorter survival time than those progression. We then analyzed expression and survival data of two with only Keap1 deficiency (Figure 2b, median survival of 22 vs other independent lung cancer studies in the GSE database 49 months; log-rank test P = 0.05, HR = 2.9, 95% CI = 1 to 8.8; (GSE3141 and GSE31210).26,27 Statistically significant association Gehan-Breslow-Wilcoxon test P = 0.04). These results suggest that between high RSPO3 expression and poor survival was found in tumors with high RSPO3 expression largely account for the poor survival of patients with Keap1-deficienct tumors that was one cohort (GSE3141, Supplementary Figure 3a; median survival of 32,33 24 vs 49 months, P = 0.001; HR = 14.7, 95% CI = 3.1–69.1) and a previously demonstrated in lung AD. fi trend existed in the other study (Supplementary Figure 3b, log- To con rm the correlation between high RSPO3 expression and fi rank test P = 0.06, Gehan-Breslow-Wilcoxon Test P = 0.04). Of note, Keap1 de ciency, we tested if the Keap1-NRF2 pathway directly the frequency of RSPO3-high tumors and the overall survival time regulates RSPO3 transcription. Analysis of the microarray data of ’ in the GSE3141 cohort are similar to those of the TCGA set, CCLE s 186 lung cancer cell lines revealed that nearly all the genes whereas the GSE31210 cohort had lower frequency of RSPO3-high that are best correlated with RSPO3 expression are in the Keap1- tumors (2% vs 11%). Nevertheless, when the data from all three NRF2 pathway (Supplementary Figure 5b). A549, one of the most commonly used cell lines for lung cancer studies, is mutated in cohorts were pooled, high RSPO3 expression was found in ~ 9% 29 (36/412) of the cases, and it was associated with poor survival Keap1, and expressed high levels of RSPO3 and LGR4 (Figure 1i, median survival of 28.4 vs 163 months, Po0.0001; (Supplementary Table 2). To test if RSPO3 expression is regulated HR = 15.7, 95% CI = 6.8–36.2). by the Keap1-NRF2 pathway, GFP-Keap1 was expressed in A549 cells, which, as expected, reduced the level of NRF2 (Figure 2c). Reverse transcription-quantitative PCR (RT-qPCR) analysis revealed High RSPO3 expression is driven by a combination of Keap1 that the mRNA level of RSPO3 in cells expressing GFP-Keap1 cells fi de ciency and demethylation of its own promoter was reduced by ~ 80% compared with GFP control cells To identify the mechanisms of RSPO3 upregulation in a subset of (Figure 2d). The expression of AKR1C2, a well-known target of lung ADs, we first searched for fusions to PTPRK, as was reported NRF2, was nearly completely repressed in GFP-Keap1 cells in colon cancer. To validate this approach, we analyzed the RNA- (Figure 2d). To further confirm these results, we used the H1944 Seq data from TCGA’s colorectal cancer cohort,28 and found that NSCLC cell line, which expresses high levels of RSPO3 and other high levels of RSPO3 occurred in 2% (4/220) of the tumors NRF2-regulated genes based on the gene expression data in CCLE. (Supplementary Figure 4). Importantly, transcripts from all four Expression of Keap1 in H1944 cells led to reduction in NRF2 level cases of RSPO3-high tumors contained recurrent fusions between with commensurate decreases in mRNA levels of AKR1C2 and exon-1 of PTPRK and exon-2 of RSPO3 (Supplementary Table 1) as RSPO3 when compared with cells expressing GFP control described previously.14 Furthermore, none of the four RSPO3-high (Supplementary Figures 6a–c). Although these results suggest tumors contained mutations in Apc or β-catenin, consistent with that overexpression of Keap1 led to reduced expression of RSPO3 their exclusivity with mutations in the Wnt pathway.14 When the in Keap1-deficient cells, it is worth noting that the level of same algorithm was applied to RNA-Seq data of the 11 lung AD recombinant Keap1-GFP was much higher than that of the samples with the highest RSPO3 expression, however, no fusion of endogenous form (Figure 2c), which may have a nonspecific RSPO3 to PTPRK or to any other gene was detected. These results effect. Previously, genome-wide analysis of gene expression suggest that in lung ADs, aberrant expression of RSPO3 was not change following knockdown (KD) of NRF2 in A549 cells was due to fusion with PTPRK. carried out by others.34 We examined this data set (GSE38332) and Next, we examined if RSPO3-high tumors were enriched for found that RSPO3 expression was decreased by 75% following the mutation of other genes commonly altered in lung AD. Initial KD of NRF2 in A549 cells. Taken together, these results indicate analysis using the OncoPrint function of cBioPortal showed 100% that the high expression of RSPO3 in A549 and H1944 cells was

120 RSPO3-norm, Keap1-norm RSPO3 100 RSPO3-norm, Keap1-def RSPO3-high, Keap1-def Keap1-m 80 (Logrank test p = 0.02) Keap1 60 ABCC2

% Surviving 40 AKR1C1 N = 162 20 AKR1C2 N = 18 N = 15 0 0 50 100 150 200 250 -Keap1-mutated -Keap1-WT -Expression high -Expression low Months of Survival

α-GFP α-Keap1 α-NRF2 150 GFP GFP-Keap1 12 3 4 5 1 2 12 12 125 -GFP- -GFP- -6824 Keap1 100 Keap1 -NRF2 -eKeap1 75 -6043 50 % Control Exp -GFP -actin 25 -5620 -actin 0 -actin AKR1C2 RSPO3

200 1.0 0.9 0.8 0.7 RSPO3-low (N = 70) RSPO3-high (N = 10) 0.6 150 0.5 (*p < 0.01, student T test) 0.5 * * 0.4 * 100 * 0.3 * * * 0.2 * 50

0.1 Fold of Control Exp

0.0 0 Mean Beta values of Methylation 56 Vehicle SF AZ SF+AZ -48 365 462 681 795 -842 -509 -398 -364 1012 1069 1103 1151 1557 1656 1945 2600 -3648 356857386477560 CpG location Relative to Transcription Start Site Figure 2. High RSPO3 expression in lung AD depends on Keap1 deficiency and RSPO3 promoter demethylation. (a) A heat map depicting the expression levels of RSPO3 and three NRF2-regulated genes vs mutation and expression status of Keap1 in TCGA’s LUAD cohort. (b) Kaplan– Meier survival analysis of patients with tumors of Keap1 deficiency and high RSPO3 expression vs those with tumors of only Keap1 deficiency and the rest of the cohort. (c) Representative WB of A549 cells stably expressing pLVX-GFP or pLVX-GFP-Keap1 with anti-GFP antibody (left panel), anti-Keap1 antibody (mid-panel) and anti-NRF-2 antibody (right panel). eKEap1: endogenous Keap1. The experiment was repeated twice. (d)RT–qPCR results of AKR1C2 and RSPO3 in GFP vector and GFP-Keap1-expressing cells. Expression data were normalized by 18S RNA and error bars are s.e.m. of three replicate experiments. (e) Representative ChIP-PCR results of three putative NRF2-binding sites located at -6824, -6043 and -5620 bp upstream of the TSS (transcription start site) of RSPO3. The antibodies used in the lanes are: 1 = anti-PolII, 2 = control IgG, 3 = anti-NRF2. Lanes 4 (no DNA) and 5 (input chromatin) are negative and positive controls, respectively. The experiments were repeated twice. (f) Comparison of methylation β value of 22 CpG’s across the RSPO3 promoter region in RSPO3-high vs RSPO3-norm tumors. (g)RT–qPCR results of RSPO3 expression in H2009 cells treated with vehicle, sulforaphane (SF, 10 μM) and azacytidine (AZ, 10 μM), and SF and AZ simultaneously (SF+AZ). Expression data were normalized by 18S RNA and error bars are s.e.m. of three replicate experiments. sensitive to loss of NRF2, consistent with the correlation between A549 cells. Overall, these bioinformatic and experimental data high RSPO3 levels and Keap1 deficiency in tumor samples. suggest that NRF2 directly binds to an upstream region of the To find if the promoter region of RSPO3 contains NRF2-binding RSPO3 promoter, which may have a major role in driving sites,35 we screened a 50-kb genomic sequence surrounding RSPO3 expression to aberrantly high levels in Keap1-deficient the transcription start site of RSPO3 and identified nine motifs lung ADs. that could potentially be bound by NRF2 (Supplementary Despite the clear relationship between Keap1 and RSPO3, Figure 7). Chromatin immunoprecipitation-PCR was carried out deficiency of Keap1 alone was insufficient to drive RSPO3 for each of the nine sites and three consecutive sites (-6824, -6043 expression as only approximately half of Keap1-deficient lung and -5620, Supplementary Figure 7) were found to be bound by ADs had high levels of RSPO3 (Figure 2a). We then searched for NRF2 in A549 cells (Figure 2e). We also examined the Encode other potential mechanism such as gene amplification, mutation Genome data using the UCSC Genome Browser (http://genome. burden, fraction of copy number altered genome and DNA ucsc.edu/index.html) and found that there is a DNaseI hypersen- methylation. No significant difference was found between sitivity site encompassing the -6820 NRF2-binding site of RSPO3 in RSPO3-high and -norm tumors in RSPO3 copy number, mutation

© 2015 Macmillan Publishers Limited Oncogene (2015) 4692 – 4701 RSPO3-LGR4 signaling in lung cancer X Gong et al 4696 burden or fraction copy number altered genome (Supplementary provide other factor(s) necessary for high RSPO3 expression. Figures 8a–d). Strikingly, RSPO3-high tumors had a significant Overall, these results support the model where NRF2 activation decrease in the level of methylation, particularly so in a region and RSPO3 promoter demethylation are the major drivers of around 1500 bp downstream of the transcription start site of RSPO3 expression in lung ADs. RSPO3 (Figure 2f). Parallel analysis of mutation, expression and methylation data of RSPO3 and Keap1 revealed that not all tumors fi High RSPO3-LGR4 signaling in lung cancer cells promotes cell with Keap1 de ciency and RSPO3 low methylation had high levels growth and migration in vitro of RSPO3 (Supplementary Figures 9a–c), indicating that additional factors are required to achieve maximal expression for RSPO3. Recently, we showed that short hairpin RNA (shRNA)-mediated KD fi fi of LGR4 or RSPO3 in A549 and H460 led to impaired Wnt Nevertheless, these ndings suggest that de ciency in Keap1 and 11 demethylation of the RSPO3 promoter are necessary but not signaling. Here we compared the growth rates of A549 sufficient for high RSPO3 expression in lung ADs. To test this expressing either an effective LGR4 shRNA (shLGR4-40) or one of three ineffective ones (shLGR4-39, -41 and -42). Cell growth in real mechanism, we used the lung AD cell line H2009, which has 36 neither Keap1 mutation (based on the lack of expression of NRF2- time was measured using the xCELLigence assay. Only the cell regulated genes) nor RSPO3 expression (Supplementary Table 2). line with the effective LGR4 shRNA (#40) grew ~ 50% slower than H2009 cells were treated with either the demethylating agent the parental cells (Figure 3a). To confirm these results, we azacytidine or the NRF2 activator sulforaphane, or both. RT-qPCR screened additional LGR4 shRNAs (a total of 15) and only found analysis showed that demethylation slightly increased RSPO3 1 (shLGR4-43) that gave a partial KD of LGR4. Cells expressing expression, whereas NRF2 activation alone had no effect shLGR4-43 showed reduced Wnt signaling to a lesser extent than (Figure 2g). Importantly, combination of demethylation and those with shLGR4-40 (Supplementary Figures 10a and b). NRF2 activation led to much higher increase (~150-fold) in Importantly, cells expressing shLGR4-43 displayed a significant expression of RSPO3 (Figure 2g), suggesting that H2009 cells decrease in cell growth, migration and invasion when compared

6 5 5 Parental Parental Parental shRNA-cont shRNA-cont 5 shLGR4-39 shLGR4-40 shLGR4-40 4 shRSPO3-63 4 shLGR4-41 shRSPO3-67 4 shLGR4-42 3 3 3 2 2 Cell Index Cell Index 2 Cell Index

1 1 1

0 0 0 0 24 48 72 96 120 144 0 24 48 72 96 120 144 0 20 40 60 80 100 120 Growth Time (Hour) Growth Time (Hour) Growth Time (Hour)

600 PCKD 250 -RSPO3 * -LGR4 +RSPO3 200 400 -actin 150 * * PCKD * 100 200 -IQGAP1 % Basal Migration Basal % # of Migrated Cells 50 -actin 0 0 0 10 100 RSPO3 (ng/ml) Vector Parental LGR4-KD IQGAP1-KD Figure 3. RSPO3-LGR4-IQGAP1 signaling regulates cell growth and migration in lung cancer cells. (a) Real-time growth curves of A549 cells with KD of LGR4 (shLGR4-40) vs parental A549 cells and three other cell lines expressing ineffective LGR4 shRNAs (#39, 41 and 42). (b) Real time growth curves of A549 cells with KD of RSPO3 (#63 and 67) vs parental A549 cells and those expressing a control shRNA. (c) Real-time growth curves of H460 cells with KD of LGR4 (shLGR4-40) vs parental H460 cells and those expressing a control shRNA. (d) Migration results of H2009 cells treated with vehicle and RSPO3 at 10 and 100 ng/ml. *Po0.05 vs control, Dunnett’s test following one-way analysis of variance (ANOVA; Po0.0001). (e) WB results of LGR4 (top panel) in parental H2009 cells (P) and those expressing a control shRNA (C) or shLGR4-40 (KD) using the antibody 7E7, and WB results of IQGAP1 (lower panel) of parental (P) and H2009 cells expressing control shRNA (C) or shIQGAP1-85 (KD). Actin was probed as loading control. (f) Migration results of H2009 cells with LGR4- or IQGAP1-KD in response to RSPO3 treatment (10 ng/ml). *Po0.05 vs parental cells, Dunnett’s test following one-way ANOVA (Po0.0001). All the experiments were repeated twice with representative data shown here. Error bars are s.e.m.

Oncogene (2015) 4692 – 4701 © 2015 Macmillan Publishers Limited RSPO3-LGR4 signaling in lung cancer X Gong et al 4697 with parental and control shRNA cells, but not to the same extent and that of E-cadherin (Figure 4e, R = − 0.20, P = 0.002, Spearman as cells of shLGR4-40 (Supplementary Figures 10c–e). Furthermore, test). Taken together, these results indicate that endogenous A549 cell lines with complete KD of RSPO3 by two independent RSPO3-LGR4 signaling not only enhances cell migration and shRNAs (#63 and 67), which we described previously11 displayed a invasion but also promotes the EMT process in lung AD cells, similar extent of decrease in the rate of cell growth (Figure 3b). which may be the underlying mechanisms for the high aggres- The decrease in cell growth was further confirmed using the MTT siveness of RSPO3-high lung ADs. (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) cell proliferation assay for both LGR4 and RSPO3 KD cells (Supple- KD of LGR4 or IQGAP1 reduced tumor growth and metastasis mentary Figures 11a and b). KD of LGR4 in H460 cells, which are in vivo also mutated in Keap1 and express high levels of RSPO3 and LGR4 We then evaluated the effect of knocking down LGR4 and IQGAP1 (Supplementary Table 2), resulted in a similar level of decrease in on tumor growth and metastasis using xenograft models of A549 cell growth (Figure 3c). Repeated efforts (twice) failed to obtain cells. In the subcutaneous model, tumors from LGR4-KD cells H460 cells with stable KD of RSPO3 using either shRSPO3-63 or showed ~ 70% reduction in weight at the end of the study when -67, whereas plenty of cells survived with vector control. These compared with those of parental or control-shRNA cells (Figure 5a results suggest that endogenous RSPO3-LGR4 signaling has an and Supplementary Figure 16a; P = 0.001, one-way analysis of important role in the regulation of cell growth in both A549 and variance; Po0.05, LGR4-KD vs parental or control shRNA cells, H460 cells. Dunnett’s test). Reduction in levels of LGR4 protein in LGR4-KD IQGAP1 has pleiotropic roles in the regulation of cell adhesion tumors was confirmed by WB (Supplementary Figure 16b). and migration in normal and cancer cells.12,13,37 We found that KD Immunohistochemical staining showed that LGR4-KD tumors of RSPO3, LGR4 or IQGAP1 in A549 cells led to significant had increased levels of E-cadherin but decreased levels of reduction in cell migration and invasion.11 Next, we tested if vimentin (Figure 5b), consistent with the reversal of EMT in RSPO3 stimulation of H2009 lung cancer cells, which express LGR4-KD cells. To determine if KD of LGR4 also affects tumor LGR4 and IQGAPs, but not RSPO3 (Supplementary Table 2), metastasis, A549 cells with control or LGR4-KD shRNA were tested would enhance cell migration. Treatment of H2009 cells with in the tail vain injection model using athymic nude mice. Loss of RSPO3 increased their migration in a dose-dependent manner LGR4 led to significant decrease in tumor formation in the lung (Figure 3d). H2009 cell lines stably expressing shRNA-targeting (1/5 for LGR-KD cells vs 5/5 for control cells, P = 0.02, Fisher’s exact LGR4 (shLGR4-40) or IQGAP1 (shIQGAP1-85), and control shRNA test). The tumors from control shRNA cells were much larger with were generated, and their KD effect was confirmed by western two animals dead before killing (Figure 5c and Supplementary blot (WB) analysis (Figure 3e and Supplementary Figures 12a and Figure 17a), whereas the only tumor formed from LGR4-KD cells b). KD of LGR4 led to a total loss of response to RSPO3 treatment was microscopic (Figure 5c and Supplementary Figure 17b). None in the migration assay, whereas parental or control shRNA showed of the mice from either group had tumors in the brain. We also increased migration as expected (Figure 3f and Supplementary tested the effect of IQGAP1 KD on tumor metastasis in luciferase- Figure 13). KD of IQGAP1 led to ~ 70% reduction in RSPO3- expressing A549 cells by in vivo imaging, which was previously induced increase in migration (Figure 3f). The residual response validated in SCID-Beige mice.39 Loss of IQGAP1 led to significant may be due to incomplete IQGAP1 KD (Figure 3e, lower panel) or decrease in lung colonization (Figure 5d). Overall, 3/7 animals with redundant function of IQGAP3 in these cells. Overall, these results control shRNA had tumors in the lung and 1/7 animal had tumors indicate that RSPO3 is able to promote cell migration through the in both the brain and lung, whereas 0/6 animals of IQGAP1-shRNA LGR4-IQGAP1 axis in lung cancer cells with normal Keap1 function. had detectable tumors (P = 0.049, Fisher’s exact test, one-tailed). Lack of lung colonization by IQGAP1-KD cells were confirmed at RSPO3-LGR4 signaling is crucial to the regulation of epithelial– necropsy. All together, these in vivo data provide further support mesenchymal transition (EMT) of RSPO3-high lung cancer cells that LGR4 and IQGAP1 have functions in the regulation of tumor It was observed that A549 cells with KD of LGR4 displayed an growth and metastasis in lung cancer cells. obvious change in cell morphology, from spread-out, mesenchymal-like to cobble-stone, epithelial-like cells (Supple- mentary Figure 14). Staining of β-catenin, a key component of DISCUSSION cell–cell adhesion, revealed that LGR4-KD cells displayed increased Dysregulation of Wnt signaling occurs in nearly every major type levels of membrane-bound β-catenin (Figure 4a, left panel) when of solid tumor with colon cancer being most frequently compared with control shRNA cells (Figure 4a, right panel). These affected.40,41 RSPOs and LGR4–6 were recently found to form a observations suggest that loss of RSPO3-LGR4 signaling trans- ligand-receptor system with critical roles in normal development formed A549 cells from mesenchymal-like to epithelial-like cells, and stem cell survival through modulation of Wnt signaling.1,4–6 that is, reversal of EMT. As the EMT process is critical to the The finding of recurrent gene fusions of RSPO2 and RSPO3 in a development of tumor metastasis,38 we then examined a series of subset of colon cancer clearly established a role of RSPO2/3 in epithelial and mesenchymal markers by WB analysis. KD of LGR4 human cancer,14 although clinicopathological characteristics of in either A549 or H460 cells led to a significant decrease in the RSPO2/3-altered colon tumors have yet to be characterized. In levels of mesenchymal markers (fibronectin, N-cadherin and NSCLCs, genetic alterations in APC and β-catenin of the Wnt vimentin) with commensurate increase in the level of the pathway have also been detected, albeit at a low frequency.42 epithelial marker E-cadherin (Figure 4b and Supplementary Here we found that RSPO3 was expressed at exceedingly high Figure 15a). The reversal of EMT markers was even more levels in a subset (~9%) of lung ADs that showed much less pronounced in cells with KD of RSPO3 (Figure 4c and favorable outcome. In contrast to the mechanism of gene fusion in Supplementary Figure 15b), consistent with the more severe colon cancer, the high RSPO3 expression in lung ADs were derived decrease in migration and invasion of these cells.11 We then from a combination of deficiency in Keap1 and demethylation of queried if levels of RSPO3 mRNA were correlated with those of the RSPO3 promoter. This is consistent with a recent publication EMT markers in primary tumors by analyzing the expression data that reported that RSPO2-EIF3E and RSPO3-PTPRK fusions were of the TCGA LUAD set. A positive correlation was found between found at a frequency of 4% (3/75) in a colon cancer cohort but expression of RSPO3 and the mesenchymal marker Snail missing in 121 primary lung cancers.43 Furthermore, we found that (Figure 4d, R = 0.46, Po0.0001, Spearmen test). In contrast, a LGR4 function promotes cell growth, migration and invasion of clear negative correlation was found between the level of RSPO3 lung cancer cells both in vitro and in vivo. These data suggest that

LGR4-KD Control shRNA

A549 H460 A549 PCKD PCKD P V 63 67 -LGR4 -Zeb1

-E-cadherin -E-cadherin

-Fibronectin -Fibronectin

-N-cadherin -N-cadherin

-Vimentin -Vimentin

-Actin -Actin

14 18

12 (R = 0.46, p < 0.0001) (R = -0.20, p =0.002) 16 10

8 14

6 12

4 10

Snail1 Exp (log2[RSEM]) 2 E-Cadherin Exp (log2[RSEM]) 0 8 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 16 RSPO3 Exp (log2[RSEM]) RSPO3 Exp (log2[RSEM]) Figure 4. RSPO3-LGR4 signaling regulates EMT in lung cancer cells. (a) Representative micrographs of confocal immunoﬂuorescence microscopy of β-catenin in A549 cells with LGR4-KD (shLGR4-40) or control shRNA. β-Catenin was stained with a mouse anti-β-catenin antibody followed by Alexa488-labeled anti-mouse antisera. (b) WB analysis of EMT markers in parental A549 and H460 cells (P) and those with KD of LGR4 (KD) or control shRNA (C). (c) WB analysis of EMT markers in parental A549 cells (P) and cells expressing vector control (V), RSPO3- shRNA #63 or #67. All the experiments were repeated twice with representative data shown here. (d) Plot of the expression of RSPO3 vs that of Snail in TCGA’s LUAD set (230 samples). (e) Plot of the expression of RSPO3 vs that of E-cadherin in TCGA’s LUAD set (230 samples).

poor survival of lung AD patients with RSPO3-high tumors was Omnibus (http://www.ncbi.nlm.nih.gov/geo). Oncogenic functions potentially due to, at least in part, aberrant RSPO3-LGR4 signaling. of RSPO2 and RSPO3 invariably depend on aberrant expression, Activation of RSPO3 transcription by NRF2 in a subset of lung either due to MMTV integration as found in mouse models of ADs is a surprise. Expression of RSPOs is tightly controlled during breast and colon cancer or due to fusion with a neighboring gene development with low levels in normal adult tissues.44 During that is highly transcribed as in human colorectal cancer.15–17,45 lung development, only RSPO2 is expressed and its function is The mechanism of RSPO3 upregulation in lung ADs is unique for essential for laryngeal-tracheal and lung morphogenesis.2,3,44 In its dependence on NRF2 activation and demethylation of its own the normal adult lung, RSPO2 is no longer expressed, whereas promoter. The three NRF2-binding site sequences upstream of the RSPO3 is found at very low levels based on analysis of gene RSPO3 promoter are not conserved in the mouse. Activation of expression data from multiple databases, including UniGene NRF2 by treatment with xenobiotics alone in normal cells did not (http://www.ncbi.nlm.nih.gov/unigene) and Gene Expression upregulate RSPO3 based on the analysis of microarray data

Oncogene (2015) 4692 – 4701 © 2015 Macmillan Publishers Limited RSPO3-LGR4 signaling in lung cancer X Gong et al 4699 E-cadherin Vimentin 1200 Parental Control-shRNA 1000 LGR4-KD ) 3 800 shRNA-cont 600 S

400 Tumor size (mm

200

0 5 LGR4-KD 10 15 20 25 30 35 Days Post-Injection S D

shRNA-cont shRNA-Cont

shIQGAP1-85

LGR4-KD Figure 5. KD of LGR4 or IQGAP1 in A549 cells reduced tumor growth and metastasis in vivo.(a) Growth curve of subcutaneous tumors of A549 cells with KD of LGR4 vs parental A549 cells and those expressing control shRNA. (b) Representative micrographs of immunohistochemistry of E-cadherin and vimentin in subcutaneous tumors derived from A549 cells with LGR4 KD or control shRNA. The stromal (S) areas are demarcated by dotted lines. (c) Representative micrographs of hematoxylin and eosin histology of lung tumors formed by A549 cells with KD Of LGR4 or control shRNA. The tumor areas (T) are demarcated by dotted lines. (d) In vivo imaging results of lung metastasis of A549-luciferase cells with KD of IQGAP1 or control shRNA. deposited in Gene Expression Omnibus. Furthermore, Keap1/NRF2 growth, but not in metastasis.47 Intriguingly, in the TCGA LUAD is mutated in 27% (48/178) of lung squamous cell carcinomas in set, high levels of Axin2, the most validated indicator of Wnt/TCF the TCGA cohort. However, none of the Keap1/NRF2-mutated signaling, were not associated with patient outcome (Supplemen- samples displayed aberrant expression of RSPO3. Moreover, only a tary Figure 18a), and expression of RSPO3 was not correlated with fraction of lung AD tumors with Keap1 deficiency and RSPO3 those of Axin2 (Supplementary Figure 18b). These observations promoter demethylation did not show high levels of RSPO3 imply that RSPO3-LGR4 most likely promotes the aggressiveness (Supplementary Figure 8). These observations indicate that the of lung ADs through enhancement of the β-catenin-independent aberrant expression of RSPO3 in selected Keap1-deficient lung pathway. This pathway, also called the non-canonical pathway, ADs depends on NRF2 activation, demethylation and other factor has a critical role in the control of cell polarity and migration of (s) found in lung ADs that are yet to be identified. normal cells and the invasion and metastasis of cancer cells.48 The much reduced survival of patients with RSPO3-high tumors Recently, we uncovered that RSPO3-LGR4 is coupled to IQGAP1 to suggests that RSPO3-LGR4 signaling enhances tumor metastasis. potentiate both the canonical and non-canonical Wnt pathways. Previously, it was reported that elevated Wnt/TCF signaling IQGAP1 has major roles in the regulation of cell adhesion and promoted aggressive metastasis of human lung cancer cells to migration of normal and cancer cells through direct modulation of the brain and bone in mouse xenograft models.46 In the K-ras actin polymerization proteins.11–13,37 Aberrant IQGAP1 activity was mouse model of lung AD, however, concomitant activation of β- suggested to promote tumor metastasis in multiple types of solid catenin with K-ras only led to increase in tumor initiation and tumors, including those of the lung.49–51 High RSPO3-LGR4 activity

© 2015 Macmillan Publishers Limited Oncogene (2015) 4692 – 4701 RSPO3-LGR4 signaling in lung cancer X Gong et al 4700 in lung AD cells may preferentially enhance the β-catenin- continuously. For the MTT assay (Cayman Chemical Company, Ann Arbor, independent Wnt pathway to promote cell migration and invasion MI, USA), cells were plated into 96-well plates at a density of 1000 cells per through modulating the roles of IQGAP1 in regulating actin well with medium mentioned above and cell numbers were determinate dynamics. Further studies will be required to dissect the exact daily for up to 10 days. Migration and invasion assays of A549 and H2009 11 mechanisms of how RSPO3-LGR4 functions to modulate the two cells were carried out as described previously. distinct pathways of Wnt signaling in lung AD cells. In conclusion, we propose that RSPO3 expression is aberrantly In vivo studies activated in a subset of lung ADs as a result of Keap1 deficiency, Six-week-old female BALB/c athymic nude mice were purchased from demethylation of its own promoter and other unknown factor(s). Charles River Laboratories (Hollister, CA, USA). For tumor growth, mice High levels of RSPO3 activate LGR4-IQGAP1 to potentiate Wnt (n = 5 in each group) were injected subcutaneously with 4 × 106 cells per signaling, leading to increases in cell growth, motility and animal in 1:1 DMEM/Matrigel (BD Biosciences, San Jose, CA, USA) into the metastasis, and therefore poor patient survival. Inhibition of right dorsal flanks. Tumor measurements were carried out blindly with a caliper at least once per week, and volumes were calculated using the RSPO3-LGR4 signaling may provide a therapeutic approach to 2 attenuate or even block the progression of lung ADs with high formula: V = 0.52 × (length) × width. Mice were euthanized when they met RSPO3 expression. the institutional euthanasia criteria for tumor size and overall health condition. The tumors were then removed and photographed. Immuno- histochemistry analysis of vimentin (Cell Signaling, #5741) and E-cadherin MATERIALS AND METHODS (Cell Signaling, #3195) was performed on paraffin sections with the Vectastain ABC-Elite kit according to the manufacturer’s instructions. Data downloading and bioinformatic analysis Experimental metastasis to the lung was induced by injecting 1 × 106 cells All data of gene expression, mutation, methylation, copy number and in 100 μl PBS through the tail vein of BALB/c athymic nude mice. Seven overall survival of TCGA’s LUAD (Provisional, 230 samples) were down- weeks following the injection, the surviving animals were killed and their loaded from the cBioPortal for Cancer Genomics (http://www.cbioportal. lungs were removed. Tissue samples were fixed in 10% buffered formalin 22 org/public-portal/), the CCLE portal (http://www.broadinstitute.org/ccle/ overnight and then washed with PBS, transferred to 70% ethanol and then 24 home) or the Broad Firehose database. Detection of gene fusions was embedded in paraffin, sectioned and stained with hematoxylin and eosin. performed with the software TopHat-Fusion on UCSC genome assembly 52,53 The entire lungs of the LGR4-KD group were scanned by hematoxylin and hg19 using default parameters. Screening of NRF2-binding sites in the eosin staining as no macroscopic lesions could be seen. For the study of promoter region of RSPO3 was performed using a published position 35 A549-luciferase cells with IQGAP1-KD, 11-week-old Fox Chase SCID-Beige weight matrix for NRF2. mice were injected with 1 × 106 cells through the tail veil at n = 7 for the control shRNA group and n = 6 for the IQGAP1-KD group. The mice were Cell lines, plasmids, shRNA constructs, stable cell line generation, imaged once per week for 6 weeks starting on week 2 post injection using recombinant proteins and tissue microarrays an IVIS-II imaging system (PerkinElmer, Waltham, MA, USA) as described.39 A549, H460 and H2009 cells were obtained from Dr Bingliang Fang at All mouse experiments were approved by the Institutional Animal Care and the M.D. Anderson Cancer Center. The cell lines were originally given by Use Committee of the University of Texas Health Science Center at Dr J Minna at the University of Texas Southwestern Medical Center. H1944 Houston. cells were from Dr J Nevins’ laboratory then at Duke University. A human Keap1 plasmid was purchased from Addgene (Cambridge, MA, USA)54 and Statistical methods Keap1 was subcloned into the vector pLVX-Ac-GFP to express Keap1 as a All statistical analyses were performed using the software GraphPad Prism5 GFP-Keap1 fusion. shRNA constructs of LGR4, IQGAP1 and RSPO3 and their (GraphPad Software Inc., La Jolla, CA, USA) unless otherwise noted. Data stable lines in A549 and H460 cells were described previously.11 Cells were are presented as means ± s.e.m. grown in Dulbecco’s modified Eagle’s medium+10% fetal bovine serum with penicillin and streptomycin. Tissue microarrays of lung ADs and normal lungs were purchased from US Biomax (Rockville, MD, USA). CONFLICT OF INTEREST Immunohistochemistry staining with LGR4 antibody 7E7 was performed as before.25 The authors declare no conflict of interest.

RT-qPCR, chromatin immunoprecipitation-PCR, ACKNOWLEDGEMENTS fl immuno uorescence and WB analysis We thank TCGA for generating the genomics data, collecting the clinical information – fl Total RNA isolation, RT qPCR of RSPO3 and AKR1C2, and immuno uores- and making them available for analysis. We also thank cBioportal for providing data 4 cence analysis were carried as we described previously. For the RSPO3 analysis and visualization tools and downloading capability. We thank Dr B Fang at induction experiment, H2009 cells were treated with azacytidine for 4 days the University of Texas MD Anderson Cancer for the cell lines A549, H460 and then induced with sulforaphane for 6 h. All qPCR probes were and H2009 cells. This work was supported in part by the Cancer Prevention purchased from Life Technologies (Grand Island, NY, USA). Chromatin and Research Institute of Texas (CPRIT, RP100678), the US National Institute of immunoprecipitation-PCR of the NRF2-binding sites were carried out as Health-NIH (R01GM102485), the Texas Emerging Technology Fund and the Janice described previously.35 Protein extraction and WB were carried out using D. Gordon endowment for bowel cancer research (to QJL), by NIH (R00LM009837, RIPA buffer plus protease inhibitors (Roche Diagnostics Corporation, TL1TR000371) and CPRIT (R1006; to JTC) and by the Texas Emerging Technology Indianapolis, IN, USA).55 Primary antibodies used are: GFP (Life Technol- Fund (to ZA). ogies, #A6455), Keap1 (Santa Cruz, Dallas, TX, USA, #sc-15246), NRF2 (Abcam, Cambridge, MA, USA, #ab62352), E-cadherin (Cell Signaling β Technology Inc., Danvers, MA, USA, #3195), -catenin (Cell Signaling REFERENCES Technology, #9562), N-cadherin (BD Transduction Laboratories, San Jose, CA, USA, #610920), Vimentin (Cell Signaling Technology, #5741), Fibro- 1 de Lau WB, Snel B, Clevers HC. The R-spondin protein family. Genome Biol 2012; nectin (BD Transduction Laboratories, #610077), Zeb1 (Cell Signaling 13:242. Technology, #3396), IQGAP1 (BD Transduction Laboratories, #610611) and 2 Bell SM, Schreiner CM, Wert SE, Mucenski ML, Scott WJ, Whitsett JA. R-spondin 2 is β-actin (Cell Signaling Technology, #4970). required for normal laryngeal-tracheal, lung and limb morphogenesis. Develop- ment 2008; 135: 1049–1058. 3 Yamada W, Nagao K, Horikoshi K, Fujikura A, Ikeda E, Inagaki Y et al. Craniofacial Cell growth and migration assays in vitro malformation in R-spondin2 knockout mice. Biochem Biophys Res Commun 2009; The xCELLigence RTCA system (Roche, Mannheim, Germany) was used to 381:453–458. monitor cell growth in real time. Briefly, 2000 cells were seeded into a 96- 4 Carmon KS, Gong X, Lin Q, Thomas A, Liu Q. R-spondins function as ligands of the well E-plate (Roche) in DMEM media with 10% fetal bovine serum (with orphan receptors LGR4 and LGR5 to regulate Wnt/beta-catenin signaling. Proc puromycin for stable cells) and the cell growth was monitored for 6 days Natl Acad Sci USA 2011; 108: 11452–11457.

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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)