Reduced Expression of PROX1 Transitions Glioblastoma Cells Into a Mesenchymal Gene Expression Subtype Kaveh M

Published OnlineFirst August 22, 2018; DOI: 10.1158/0008-5472.CAN-18-0320

Cancer Tumor Biology and Immunology Research

Reduced Expression of PROX1 Transitions Glioblastoma Cells into a Mesenchymal Gene Expression Subtype Kaveh M. Goudarzi1, Jaime A. Espinoza1, Min Guo2, Jiri Bartek1,3, Monica Nister 2, Mikael S. Lindstrom€ 1, and Daniel Hagerstrand€ 2

Abstract

The homeodomain transcription factor PROX1 has been ment with a CDK2 inhibitor subsequently decreased PROX1 linked to several cancer types, including gliomas, but its expression, which was paralleled by decreased SOX2 levels. functions remain to be further elucidated. Here we describe The THRAP3 protein was a novel binding partner for PROX1, a functional role and the prognostic value of PROX1 in and suppression of THRAP3 increased both transcript and glioblastoma. Low expression of PROX1 correlated with poor protein levels of PROX1. Together, these findings highlight overall survival and the mesenchymal glioblastoma subtype the prognostic value of PROX1 and its role as a regulator of signature. The latter finding was recapitulated in vitro, where glioblastoma gene expression subtypes, intratumoral hetero- suppression or overexpression of PROX1 in glioma cell cul- geneity, proliferation, and cell-cycle control. tures transitioned cells to a mesenchymal or to a nonmesenchymal glioblastoma gene expression signature, respectively. Significance: These findings demonstrate the role and PROX1 modulation affected proliferation rates that coincided prognostic value of PROX1 in glioblastomas; low PROX1 with changes in protein levels of CCNA1 and CCNE1 as well as levels correlate with a mesenchymal gene expression the cyclin inhibitors CDKN1A, CDKN1B, and CDKN1C. Over- subtype and shorter survival in glioblastoma tumors. expression of SOX2 increased PROX1 expression, but treat- Cancer Res; 78(20); 5901–16. 2018 AACR.

Introduction the human central nervous system, or alternatively from differ- entiated cells of different lineages as for example astrocytes or Glioblastoma represents the most common and aggressive oligodendrocytes (7). The human glioblastoma transcriptome primary brain tumor type in adults (1). Its intrusive growth and has been found to resemble normal outer radial glial cells and the plastic nature of the tumor make complete surgical resection intermediate progenitors (8). In support of this, it was recently impossible and the tumor cells prone to evade chemo- and shown that glioblastoma initiation is associated to aberrant radiation therapy (2). Stem cell regulatory pathways are shown reactivation of a normal developmental program in the brain activated in gliomas supporting self-renewal, tumor mainte- (9). Therefore, a better understanding of developmental pathways nance, and survival under stress (3). Furthermore, glioblastomas and their involvement in glioblastoma is thought to lead to new are very heterogeneous on an intratumoral level, composed of therapeutic possibilities. tumor cells displaying different gene expression signatures con- Prospero-related homeobox 1 (PROX1) is a transcription stituting the different glioblastoma tumor subtypes (4). Thus, a factor that mediates cell fate decisions of neuroblasts, as glioma stem-like phenotype, cell motility, and tumor cell het- reviewed in ref. 10. In several instances, PROX1 has been shown erogeneity are considered signiﬁcant hurdles to overcome for to play an active role in cancer. For example, PROX1 suppresses developing new treatment against these tumors (5, 6). the growth of neuroblastoma (11), whereas it enhances colo- Glioblastoma may arise from adult neural stem cells or multi- rectal cancer progression as a b-catenin/TCF/LEF target gene potent neural progenitor cells that persist in proliferative niches in contributing to a transition from early to a dysplastic stage (12). Prox1 regulates the number of cancer stem cells, by promoting 1SciLifeLab, Division of Genome Biology, Department of Medical Biochemistry cell proliferation and thereby expanding the cancer stem cell and Biophysics, Karolinska Institutet, Stockholm, Sweden. 2Cancer Center population in intestinal adenomas and colorectal cancer after Karolinska, Department of Oncology-Pathology, Karolinska Institutet and activation of the Wnt pathway (13). In human astrocytic brain 3 þ Karolinska University Hospital at Solna, Stockholm, Sweden. The Danish Cancer tumors, the percentage of PROX1 cells has been shown to Society Research Centre, Copenhagen, Denmark. increase with tumor grade (14). Furthermore, level of PROX1 Note: Supplementary data for this article are available at Cancer Research predicted survival in grade II gliomas, where a percentage of þ Online (http://cancerres.aacrjournals.org/). PROX1 cells over 10% correlated with worse outcome (15). Corresponding Author: Daniel Hagerstrand,€ Cancer Center Karolinska, Depart- Moreover, PROX1 has been proposed as a novel pathway- ment of Oncology-Pathology, Karolinska Institutet and Karolinska University speciﬁc prognostic biomarker for high-grade astrocytomas Hospital, Stockholm 17176, Sweden. Phone: 46-0-8-517-705-85; E-mail: (16). Here we sought further understanding of the role of [email protected] PROX1 in glioblastoma by analyzing publicly available gene doi: 10.1158/0008-5472.CAN-18-0320 expression data from tumor samples and by performing in vitro 2018 American Association for Cancer Research. experiments.

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Materials and Methods fection of plasmid DNA and siRNA, respectively, according to the Cell culture manufacturers instructions. Glioblastoma cell lines U-343 MG, U-343 MGa, U-343 MGa Publicly available gene expression data Cl2:6, U118MG, U178MG, U373MG, and U1242MG have pre- The Cancer Genome Atlas (TCGA) data from 539 glioblastoma – viously been characterized (17 21) and references therein. The samples, published in 2013 (25) was downloaded from UCSC high-grade glioma cultures 4, 11, and 18 were initially character- University of California Santa Cruz Xena portal (www.xenabrow ized based on gene expression and phenotypes (22), and have in ser.net). Data for glioblastoma gene expression based subtypes other studies also been referred to as U2975, U2982, and U2987 and patient survival were downloaded from cBioPortal for Cancer (23), respectively. For unity, these cultures are referred to as Genomics at http://cbioportal.org and merged with the expres- U2975(4), U2982(11), and U2987(18) in this study. The cell sion data. Samples categorized as G-CIMP or taken from relapse lines and cultures above were retrieved from authors' lab stocks tumors were removed yielding a final set of 375 tumors. Gene (22) and subsequently cultured for less than 3 months. All cell expression data for cell cultures was downloaded from the Cancer fi lines were maintained in DMEM (Thermo Fisher Scienti c) with Cell Line Encyclopedia (CCLE) (26) database at www.broadin – 10% FBS, 2 mmol/L L-glutamine, and penicillin streptomycin at stitute.org/ccle. Cell cultures described to be derived from glio- > 37 C with 5% CO2, and 5% O2 and 95% humidity. The other blastomas were selected, which generated a set of 45 cell cultures. glioma cell lines (U87MG, U251MG, M059J, and M059K), as Gene expression data for 48 glioblastoma cell cultures was down- well as the osteosarcoma line U2-OS and colon cancer line loaded from the human glioblastoma cell culture (HGCC) portal SW480 were purchased from ATCC and cultured per standard at www.hgcc.com (27). guidelines for less than a month. SOX2 and YFP (control) overexpressing U-343 MG cultures were generated by lentiviral trans- Survival analysis of patients with glioblastoma duction with pLEX-Blast-V5-SOX2 and pLEX-Blast-V5-YFP by Analyses of overall and disease-free survival were conducted methods previously described (24). To confirm the relationship using data available for patients with glioblastoma, published in between the U-343 clones they were subjected to STR profiling at 2008 (28), extracted from http://cbioportal.org and patients NGI-Uppsala, SciLifeLab, Uppsala University, using the Amp- with glioma from http://gliovis.bioinfo.cnio.es/. Statistical ana- FISTR Identifiler PCR Amplification Kit (Thermo Fisher; Supple- lysis of the data was performed using an unpaired two-tailed t-test mentary Table S1). The cultures used in this study were screened assuming normal distributions (Prism 6.0; Graphpad software for Mycoplasma using the MycoAlert Mycoplasma Detection Kit Inc.). Kaplan–Meier survival curves were plotted for patients (LT07-218; Lonza). Stock solution of CVT-313 (Santa Cruz) was grouped according to PROX1 expression levels above or below prepared by dissolving powder in DMSO, stored at 20C, and 1, and THRAP3 above or below 0.3. Log-rank (Mantel–Cox) tests used by dissolving stock solution in cell culture media and were used to determine differences between survival curves. incubating with cells at concentration and time indicated. Gene set enrichment analysis Lentiviral transduction and transfection of plasmids or For the gene set enrichment analysis (GSEA; ref. 29), we used siRNA constructs the software on the GenePattern online server (www.genepattern. To generate a PROX1 overexpressing cell culture, U-343 broadinstitute.org) module version 18 and the Molecular Signa- MG-PROX1, purified lentivirus (Lentifect) for PROX1 transduc- tures Database (MSigDB; ref. 30) version 6.0. Overlaps of PROX1 tion into U-343 MG was purchased (PROX1 containing virus correlated gene sets were computed with the following categories LPP-F0925-Lv105-100-S and negative control virus LPP-NEG- in MSigDB collections using the default setting (FDR q value LV105-100-C; GeneCopoeia), and used according to manufac- below 0.05). For GSEA of the CCLE data for 45 glioblastoma cell turer's instructions. To study PROX1 suppression in U-343 MGa, cultures, we divided the cultures into two groups based on the the cell line U-343 MGa-shPROX1 was generated using lentiviral expression of PROX1. To determine at what cutoff to divide vectors against PROX1 (TRCN0000232123; Sigma-Aldrich) or the sample data into two groups according to PROX1 expression, unrelated control shRNA targeting GFP (TRCN0000072178, the cultures were first ranked per expression level of PROX1. The clonetechGfp_438s1c1, previously described; ref. 24). Virus pro- t tests were then performed between high and low PROX1 expres- duction was performed by calcium-phosphate-mediated cotrans- sion in the samples for all the genes. The t test was performed fection of HEK 293T cells with packaging plasmids (MISSION for all permutations starting with the two cultures with the Lentiviral Packaging Mix, SHP001; Sigma-Aldrich). Twenty-four highest PROX1 versus the rest, the three cultures with the hours after transfection, the different supernatants were collected highest PROX1 level versus the rest, and so on. For each two times with 24-hour intervals, filtered and then used to infect permutation, the t-test significance was determined for all genes U-343 MGa cells cultured under optimal conditions. Lentiviral and internally compared with the P-value–based rank position transductions of glioblastoma cells were conducted at MOI of 1 to of PROX1. The cutoff for high versus low PROX1 expressing 2. The cells were analyzed and selected in the presence of 1 mg/mL cultures was set to where PROX1 had the highest relative puromycin for 72 hours or until all nontransduced control cells P-value ranking compared with other genes. In other words, were killed. Transduced cells were kept in culture for 3 days, the cut-off was set to where PROX1 had the lowest relative P prior to experiments. For RNAi experiments SMARTpool mixes, value compared with other genes. We found the optimal cutoff ON-TARGETplus siRNA (GE Healthcare Dharmacon), were used when the cultures were divided into 25 low versus 20 high to target PROX1 (L-016913-00-0005), SOX2 (L-011778-00- PROX1 expressing cultures where PROX1 was the 11th most 0005), or THRAP3 (L-019907-00-0010) or nontargeting control significant gene to distinguish the samples. We then used the 25 pool (D-001810-10-05) according to the manufacturer's instruc- low versus 20 high PROX1 expressing culture division for tions. Lipofectamine 2000 (Thermo Fisher Scientific), and RNAi- further GSEA analyses. The analyses were run with 1000 per- MAX (Thermo Fisher Scientific) reagents were used for the trans- mutations, phenotype as permutation type, collapsed data set,

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and the AFFYMETRIX chip platform file option. In this case, we centroid genes previously described by (32), where each subtype investigated the gene set databases that included all subcate- was described by a set of 210 centroid genes with either high or gories including positional, curated, motif, computational, low expression as compared with the others. For instance, in our gene ontology, oncogenic signatures, immunologic signatures, analysis corresponding to U-343 MG-PROX1 versus U-343 MG and hallmarks. for the mesenchymal subtype, 35 of 128 genes with available measurements were increased more than one-fold, whereas 12 of RNA purification and quantitative real-time PCR 33 genes decreased more than one-fold. This resulted in a Total cellular RNA was extracted from 70% to 80% confluent rescaled index of 42 for the mesenchymal subtype, when cultures using PureLink Kits (Ambion RNA extraction products) 100 is full negative change of subtype, 0 is neutral, and 100 according to manufacturer's instructions (Thermo Fisher Sci- is full positive change. entific). RNA concentrations were measured with a NanoDrop spectrophotometer and samples were stored at 80 C. For Cell proliferation assay gene expression analysis, real-time quantitative RT-PCR (qRT- Cell proliferation assay was performed by cell counting using PCR) was performed. The Power SYBR Green RNA-to-CT 1-Step Countess automated cell counter (Thermo Fisher Scientific). For Kit (Thermo Scientific) was used according to the manufac- proliferation analysis, cells were seeded in 12-well plates and the turer's instructions in conjunction with a 7500 StepOnePlus total cell number was determined at the indicated time points. The Real-Time PCR system (Thermo Scientific). GAPDH was used as experiments were repeated three times using triplicate samples. the internal standard reference. Gene expression levels were Statistical analysis of the data was performed using an unpaired calculated using the relative DDCt method. The primers used for Student two-tailed t test. P values <0.05 were considered statis- qRT-PCR are listed in Supplementary Table S2. Statistical anal- tically significant. ysis of the data was performed using an unpaired Student two- tailed t test (Prism 6.0; Graphpad software Inc.). P < 0.05 was Immunoblotting, coimmunoprecipitation, and mass considered statistically significant. spectrometry analysis For detection of specific antigens the following antibodies RNA sequencing and gene expression analysis were used; PROX1 (R&D Systems; AF2727), PROX1 (Abcam; RNA quality was assessed by electrophoresis on the Tapesta- ab37128), p53 (Santa Cruz; sc-6243), p53 (Santa Cruz; tion instrument (Agilent Technologies) and samples with a RIN sc-6243-G), p57 Kip2 (Abcam; ab75974), P21 (Abcam; ab7960), score above 8 were used in further analysis. RNA library prep- P21 (Santa Cruz; sc-397), cyclin B1 (Santa Cruz; sc-245), cyclin aration, sequencing, raw data processing, and quality control D1 (Santa Cruz; sc-8396), cyclin E (Abcam; ab7959–1), cyclin A2 were performed at the National Genomics Infrastructure at (Abcam; ab16726), Cdk2 (Santa Cruz; sc-6248), TRAP150 (Santa Science for Life Laboratory, Stockholm, Sweden). Reads were Cruz; sc-48779), b-actin (Sigma-Aldrich; clone AC15), SOX2 mapped using Tophat (2.0.4.) and FPKM values were generated (EDM Millipore; AB5603), GFAP (Abcam; ab10062), and FN1 with Cufflinks (2.1.1.). For further gene expression based anal- (BD Biosciences; 610078). For analysis of detergent-soluble pro- yses, we filtered transcripts from the expression data (FPKM teins, we used 0.5% Nonidet P-40 lysis buffer with complete pro- values) that were present for the CCLE gene-centric RMA-nor- tease and phosphatase inhibitors (PhosSTOP, cOmplete ULTRA; malized Affymetrix data, retaining about 12,042 genes. Signif- Roche). Analysis of detergent-soluble proteins, coimmunopre- icant difference in expression was calculated in Prism applying a cipitation (co-IP), and IB were conducted as described (33). For t test to two biological groups: (i) U-343 MG-control versus co-IP ("large-scale") followed by mass spectrometry, the cells were U-343 MG-PROX1 with two replicates in each group and harvested and prepared according to the nuclear complex co-IP kit (ii) U-343 MGa-control versus U-343 MGa-shPROX1 with three instructions (Nuclear Complex Co-IP Kit; Nordic Biolabs), using replicates in each group. Volcano plots were generated by using the high stringency buffer option (Active Motif). For the initial co- genes with FPKM values over 0.01, a two-fold change in expres- IP in U2-OS cells, we used an antibody against the FLAG tag (Flag sion and a P value below 0.05. Venn diagrams were generated M2, F1804; Sigma-Aldrich). Immunoprecipitates were separated using GeneVenn (http://genevenn.sourceforge.net) to identify by SDS-PAGE (NuPAGE Novex 4–12% Bis-Tris Protein Gels; Life PROX1 induced or suppressed genes in an extended list only Technologies), the gels stained with colloidal CBB Kit (Life with more than two-fold difference in expression between Technologies), bands unique to the PROX1-FLAG antibody lane groups of replicates, where the average of either replicate group were cut out, and identified by mass spectrometry (LC/MS-MS; was not 0. The PROX1-induced and -repressed genes were Alphalyse A/S; Denmark). Protein samples were reduced and investigated at MSigDB (30) to identify enriched gene sets. alkylated with iodoacetamide, that is carbamidomethylated, and RNA-seq data have been deposited at EBI ArrayExpress database trypsin digested. Peptides were concentrated on a ZipTip micro- with the accession number E-MTAB-6991. To generate gene purification column and eluted onto an anchorchip target to be expression heat maps, the Heat map module at the GenePattern used for analysis on a Bruker Autoflex III MALDI TOF/TOF site was used (31). instrument. MALDI MS-MS was performed on 15 peptides for peptide fragmentation analysis. Peptide tolerance was set to 60 Glioblastoma gene expression based subtype analysis ppm (one miscleavage allowed). The MS and MS-MS spectra were To assess changes of glioblastoma gene expression subtypes combined and Mascot software version 2.2.03 was used. The due to PROX1 overexpression in U-343 MG-PROX1 or suppres- database NRDB was used for protein identification. sion in U-343 MGa-shPROX1, we counted the number of subtype defining genes whose expression were altered in our RNA-seq Immunofluorescence staining experiment following modulation of PROX1. To assess the change Procedures for immunofluorescence staining and microscopy of each gene expression subtype we used the subtype defining analysis have previously been published (33).

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Results and 150 grade IV gliomas from the GlioVis portal (http://gliovis. Low PROX1 mRNA level in glioblastoma tumors correlates bioinfo.cnio.es/) to assess PROX1 expression levels (34). PROX1 with mesenchymal gene expression subtype and with shorter transcripts were signiﬁcantly less in grade IV tumors as compared survival with grade II (P < 0.0001) and III (P < 0.0001) tumors (Fig. 1A). Because PROX1 has been shown to have diagnostic and prog- In a survival analysis, patients with glioma with high PROX1 nostic value in gliomas, we utilized gene expression datasets to expression levels survived longer, with a median survival of further explore this. First, we investigated a combined low- and 64.6 months versus 40.5 for the subgroup with low PROX1 high-grade glioma dataset composed of 226 grade II, 244 grade III, expression levels (P < 0.0001; Fig. 1B). To investigate the

Figure 1. Comparison of PROX1 expression in glioma tumors of different grades and PROX1 correlation with survival and gene expression subtype. A, Comparison of PROX1 transcript levels in grade II to IV gliomas in a combined TCGA dataset (226 grade II, 244 grade III, and 150 grade IV gliomas; http://gliovis.bioinfo.cnio.es/). B, Kaplan–Meier curves showing that grade II to IV gliomas with high PROX1 levels had longer survival: median 67.5 months versus 34.9 months for the subgroup with low PROX1 (P < 0.0001). C, Analysis of PROX1 expression z-scores cutoffs (2andþ2 threshold) in TCGA data for 206 patients with glioblastoma (http://cbioportal.org). Values between 0.8 and 1.3 for overall survival, and 0.9 and 1.3 for disease-free survival yielded significant results for differential survival. D and E, Kaplan–Meier curves showing significantly higher disease-free and overall survival for PROX1 expression z-score > 1. Patients with a PROX1 expression higher than z-score 1 had significantly higher overall survival (log-rank test, P ¼ 0.00115), with the median survival 33.6 months as compared with 12.3 months for those with z-score 1 in the dataset. F, Distribution of PROX1 expression values for 375 patients with glioblastoma with available molecular subtypes in TCGA. There were no recurrent tumors included among the cases. Cases were divided into four equally sized groups based on PROX1 high to low expression values as indicated in the figure. One to 94 with high, 95 to 188 medium high, 188 to 282 medium low, and 283 to 375 low PROX1 expression. Corresponding subtypes are color-labeled in blue (proneural, PN), beige (neural, N), black (classical, CL), and red (mesenchymal, MES). G, Distribution of the percentage of each glioblastoma subtype between patients with high, medium high, medium low, and low expression of PROX1, according to the groups 1, 2, 3, and 4 in F, respectively. H, Scatter plot showing significantly lower PROX1 expression (z-score) in mesenchymal subtype compared with proneural (P < 0.0001) or classical subtype (P < 0.0001) in patients with glioblastoma from the same TCGA dataset as used in F.

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prognostic value of PROX1 exclusively in glioblastomas, we U-343 MGa Cl2:6 and U-343 MGa 31L) were originally derived extracted gene expression data available for 206 patients with from one glioblastoma tumor, and have previously been charac- glioblastoma from TCGA at http://cbioportal.org. Survival differ- terized (17, 19). The U-343 MG cells express fibronectin (FN1) ences for different PROX1 expression z-score cutoffs (2to but not glial fibrillary acidic protein (GFAP), and display a bipolar þ2 thresholds) were analyzed and values between 0.8 and 1.3 fibroblastic cell shape. The U-343 MGa cells however express for overall survival, and 0.9 and 1.3 for disease-free survival GFAP but not FN1, and they display a polygonal morphology. yielded significant differential survival (Fig. 1C). Specifically, the Corresponding levels of glioma cell protein markers SOX2, GFAP, median overall survival for patients with a PROX1 expression and FN1 were also determined in the panel of cell lines by higher than z-score 1 was 33.6 months as compared with 12.3 immunoblotting. The astroglial stem cell related proteins SOX2 months for the subgroup with lower expression (16 vs. 190 and GFAP were present in all the PROX1 expressing cells, whereas patients; P ¼ 0.0015), whereas the median disease-free survival FN1 was expressed in all PROX1 negative cultures, except U-343 for patients with a PROX1 expression higher than z-score 1 was MGa 31L, and U87MG (Fig. 2A). Thus, PROX1 is differentially 16 months compared with 6.5 (13 patients vs. 149 patients; expressed in glioma cell lines and is often coexpressed with SOX2 P ¼ 0.0118; Fig. 1D and E). To investigate the correlation and GFAP (Fig. 2A). The relatively high expression of FN1 in U- between PROX1 expression and molecular subtypes in glioblas- 343 MG, and high expression of GFAP in U-343 MGa cultures was toma, we explored a TCGA dataset (25) containing 375 patients confirmed by qRT-PCR (Fig. 2C). Given that the two cell lines U- with glioblastoma with available subtype calling. We divided the 343 MG and U-343 MGa are derived from the same tumor but patients into four equally sized groups based on PROX1 high to differ in levels of PROX1 protein (Fig. 2A), we chose these two for low expression values (Fig. 1F). We found that the group with further analysis of PROX1 function. Using lentivirus, we generated highest PROX1 expression, patients 1 to 94 (group 1), was mostly stable cell lines with either PROX1 overexpression or shRNA- represented by tumors of classical and proneural subtypes (Fig. mediated PROX1 suppression. In U-343 MG-PROX1 or MGa- 1G). On the contrary, in tumors with lowest PROX1 transcript shPROX1, we achieved stable overexpression or suppression of levels, patients 283–375 (group 4), mesenchymal and neural PROX1 at the mRNA and protein level, respectively (Fig. 2D and subtypes were the most abundant. Specifically, significant lower E). Experiments using double-immunofluorescence staining have PROX1 expression (z-score) was found in mesenchymal subtype previously shown the existence of GFAP and PROX1 double compared with proneural (P < 0.0001) or classical subtype positive cells in grade IV brain tumors (14). To investigate if (P < 0.0001; Fig. 1H). Together, the proneural and classical have PROX1 is a marker or driver of the U-343 MGa phenotype defined overall a higher PROX1 expression, whereas neural and mesen- by high GFAP expression, we performed qRT-PCR analysis. We chymal cases have lower PROX1 expression levels. Thus, low found increased GFAP mRNA levels upon overexpression of PROX1 expression defines a subset of patients enriched for mes- PROX1 in U-343 MG, and decreased levels in U-343 MGa enchymal and neural glioblastoma subtypes that display shorter upon suppression of PROX1 (Fig. 2F). Accordingly, immunoflu- disease free and overall survival. Of note, the neural subgroup has orescence staining and immunoblotting of U-343 MGa-shPROX1 been associated with contamination of normal neuroepithelial samples revealed reduced GFAP protein (Fig. 2G and H). Similar cells and nontumor-specific expression signature and should to U-343 MGa, the high-grade glioma culture U2987(18) therefore be considered with caution (35). expresses GFAP endogenously (22). We found that a transient suppression of PROX1 in this culture was sufficient to decrease Expression analysis of PROX1 in different glioblastoma cell GFAP transcript levels as determined by qRT-PCR (Fig. 2I). Taken lines identifies coexpressed genes and functional implications together, PROX1 correlates with and regulates GFAP expression in To assess gene expression differences between glioblastoma human glioblastoma cells, thus could be a driver of the U-343 cultures with high or low PROX1 expression levels, we performed MGa phenotype. GSEA for gene expression data from 45 glioblastoma cell lines in the CCLE (26). Twenty-five cultures were defined to contain high Global gene expression analysis by RNA-seq upon PROX1 PROX1 levels versus 20 with low. A ranked list of genes was overexpression or suppression in glioblastoma cells generated and queried against gene sets deposited at MSigDB To gain a more comprehensive understanding of PROX1 as a (29). A total of 137 gene sets were significantly enriched for transcription factor in glioblastoma, gene expression analysis was glioma cultures with relatively high PROX1 expression and 71 conducted by RNA sequencing. We analyzed differential gene gene sets for low PROX1 expressing glioblastoma cultures (Sup- expression upon PROX1 overexpression in U-343 MG or supplementary Table S3). pression in U-343 MGa cells (Fig. 3A). A total of 394 genes were significantly upregulated more than two-fold and 139 down- Analysis of protein and gene expression in glioma cell cultures regulated significantly more than two-fold after PROX1 over- and generation of stable cell lines to assess PROX1 function expression. In the same way, 137 versus 93 genes were altered To evaluate PROX1 expression we compared protein levels in upon PROX1 suppression (Fig. 3A). For further analyses, we a set of glioma cancer cell cultures including the clonal model compared intersectional genes between PROX1 overexpression U-343 (19) by immunoblotting and immunofluorescence stain- and suppression using an extended gene list applying only a cut- ing. We found that U-343 MGa 31L, U-343 MG, U87MG, U2982 off of two-fold expression change. We selected 1,472 genes whose (11), U2975(4), M059J, and M059K had undetectable levels of expression was increased more than two-fold upon PROX1 over- PROX1 protein, whereas it was readily detected in U-343 MGa expression in U-343 MG and 733 that were decreased more than Cl2:6, U-343 MGa, U373MG, U251MG, and U2987(18) (Fig. 2A two-fold upon PROX1 suppression in U-343 MGa. From these, we and B). PROX1 also displayed a nuclear localization as previously identified 237 overlapping genes, which we defined as PROX1- described in the positive control colorectal cancer cell line SW480 induced genes (Fig. 3B). Conversely, the expression of 855 genes (Fig. 2B; ref. 12). U-343 MG and U-343 MGa (and its derivatives increased more than two-fold upon PROX1 suppression in U-343

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Figure 2. Expression analysis of PROX1 in different glioma sample collections and generation of stable cell lines to assess PROX1 function in glioblastoma. A, Screening of glioma cell lines for PROX1, as well as SOX2, GFAP, and FN1 protein levels. Immunoblot of b-actin protein was used as loading control. B, PROX1 protein was detected by immunoﬂuorescence staining in U-343 MGa Cl2:6, indicating a nuclear localization, whereas in U-343 MG and U87MG the signal was undetectable. Colorectal cancer cell line SW480 was used as a positive control for PROX1 staining. C, Comparisons of endogenous GFAP and FN1 transcript levels in U-343 MG and U-343 MGa clones. D, Analysis of PROX1 mRNA levels in U-343 MG-PROX1 and U-343 MGa-shPROX1 as determined by qRT-PCR. E, Analysis of PROX1 protein levels in U-343 MG-PROX1 and U-343 MGa-shPROX1, as determined by immunoblotting. F, qRT-PCR analysis indicating enhanced GFAP mRNA expression in U-343 MG-PROX1 line as compared with U-343 MG and suppression of PROX1 in U-343 MGa-shPROX1 cells. G, Immunoﬂuorescence staining of GFAP (green) in U-343 MGa-shPROX1 cells shows a reduction of the protein. Cell nuclei were stained using DAPI (blue). H, Immunoblots indicating decreased GFAP protein in U-343 MGa-shPROX1 as compared with U-343 MGa, where U2-OS lysate was used as negative control. I, qRT-PCR data indicating a decline in expression of GFAP mRNA in U2987(18) cultures transiently transfected with siPROX1 oligomers. All gene expression analyses were normalized to GAPDH.

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Figure 3. Global gene expression analysis by RNA-seq upon PROX1 overexpression and suppression. A, Volcano plots depicting differential gene expression upon PROX1 overexpression or suppression in U-343 MG or U-343 MGa, respectively. B, Venn diagram illustrating 237 genes, the expression of which was increased in U- 343 MG-PROX1 and decreased in U-343 MGa-shPROX1 more than two-fold (PROX1 induced). C, Venn diagram depicting the number of genes with decreased expression in U-343 MG-PROX1 and increased expression in U-343 MGa-shPROX1 (PROX1 repressed). D, Top 10 enriched GO terms (C5: biological process) for the 237 induced genes identified by double filtering in B. Red line, P value of 0.05 as visual threshold. E, Overlap of these 237 genes found in B, with hallmark gene categories processed by GenePattern. F, GO analysis for the 286 gene intersect found in C. G, Overlaps of genes identified in C, with the hallmark gene categories (H and I). Thirty-eight genes intersect found to be positively regulated by PROX1 and correlated in TCGA, and 93 PROX1 repressed genes found to be correlated in TCGA data. J, Depicted here are GO analyses and identification of overlaps with hallmark genes categorized for the combined gene list of the overlapping genes from H and I.

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MGa, and 1199 genes decreased more than two-fold upon PROX1 POU3F2, and GAP43 (Fig. 4C). Moreover, the intersect genes that overexpression in U-343 MG. Out of these, 286 overlapped were upregulated more than two-fold were enriched for gene sets and are from here on referred to as PROX1-repressed genes including the "classical" and "proneural" subsets, "neural devel- (Fig. 3C). opment" and "upregulated in glioblastoma cells with capacity to From a database (MSigDB) analysis of the 237 PROX1-induced form neurospheres" (Supplementary Table S4). We also noted genes, we found enrichment of GO terms including neurogenesis that FABP7, PTPRZ1, and GAP43 were among the top stemness and regulation of cell proliferation (Fig. 3D) and overlaps with signature genes in the single-cell gene expression data described hallmark gene categories (Fig. 3E). Similarly, results from the by Patel and colleagues (4). Upon analysis of the Patel and analysis performed for the PROX1-repressed genes are presented colleagues stem cell signature genes, we found that PROX1 in Fig. 3F and G. Of note, both PROX1-induced and PROX1- increased the expression level of 27 of the 44 genes (61%) with repressed gene sets were enriched for hallmark genes implicated in available expression values, highlighting the connection between epithelial to mesenchymal transition. PROX1 and stemness regulation in glioblastoma (Fig. 4D). To To assess the relevance of our identified PROX1-induced and investigate PROX1 expression pattern at a single-cell level, we -repressed genes in vivo, we performed further analysis of these by analyzed the single-cell RNA-seq data available for five glioblas- comparing them with PROX1 correlated genes in glioblastoma tomas (4). Because a PROX1 expression measurement was not TCGA expression data (Fig. 3H and I). The Venn diagram available in the dataset, we developed a PROX1 proxy profile to in Fig. 3H presents that 16% (38 of 237) of PROX1-induced assess PROX1-related expression patterns. Specifically, we calcu- genes identified here overlap with the top 2,000 genes positively lated a proxy profile based on the average expression of the top five correlated to PROX1 expression in TCGA glioblastoma cases. PROX1-regulated genes from the Patel and colleagues stemness Conversely, 32% of PROX1-repressed genes overlapped with the signature, composed of SPARCL1, PTPRZ1, FABP7, GLUL, and top 2,000 negatively correlated to PROX1 expression (Fig. 3I). GO GAP43.Wefiltered and ranked the top 100 positively correlated terms and hallmark gene categories were also identified for a genes to the proxy profile in the Patel and colleagues dataset. In merged list of the intersecting genes from Fig. 3H and I, as each of the tumors we observed cell populations with varying illustrated in Fig. 3J. Collectively, our analysis of RNA-seq data expression levels of genes positively correlated with the PROX1 facilitated the identification of PROX1-regulated functional gene proxy (Fig. 4E). By analyzing single-cell data, we demonstrate cell networks, including those controlling neurogenesis, regulation of heterogeneity within tumors regarding genes correlated to the cell proliferation, and epithelial to mesenchymal transition. PROX1 proxy profile. Furthermore, we found that 71% of genes with a fold change more than 2 (30 of 42; among the top 100) PROX1 regulates gene expression-based subtypes in could be upregulated by PROX1 overexpression, based on the glioblastoma RNA-seq analysis of U-343 MG-PROX1 (Fig. 4E). Together, the Because PROX1 expression in our survey of TCGA data corre- combined analysis of functional effects of PROX1 in cell cultures, lated with glioblastoma subtypes (Fig. 1G and H), we used our coexpression in multiple gene expression datasets, including both gene expression data to assess its regulation of these subtypes. We bulk tumor and single-cell measurements, describes PROX1 as a calculated a support index for each signature by counting the regulator of glioblastoma tumor evolution that distinguishes number of classifier genes (32) with altered expression for each subpopulations of cells in heterogeneous tumors and whose loss corresponding subtype. We found that PROX1 overexpression of expression enables a switch from a nonmesenchymal to a decreased the support index for mesenchymal subtype and mesenchymal phenotype. increased it for nonmesenchymal subtypes in U-343 MG cells. Upon a Chi-square test the change of genes supporting a mesen- PROX1 overexpression induced cell-cycle regulators and chymal subtype was found significant with P < 0.000095 (Fig. 4A; increased cell proliferation þ þ Supplementary Fig. S1). Conversely, PROX1 suppression signif- It has previously been shown that SOX2 /GFAP glioblastoma icantly (Chi-square, P-value 0.017) increased the mesenchymal cultures have higher rates of proliferation and tumorigenicity, as support index in U-343 MGa cells (Fig. 4B; Supplementary Fig. compared with those with mesenchymal profile characterized by S1). Thus, PROX1 modulation altered the glioblastoma gene high expression of FN1 (22). We explored data from the HGCC expression subtype in cell cultures, where overexpression shifted biobank and found that high expression of PROX1 correlated with cells into a nonmesenchymal and suppression into a mesenchy- high tumorigenicity of the HGCC cultures as previously assessed mal subtype. by intracranial injection into NOD-SCID mice (Fig. 5A; ref. 27). To pinpoint a system independent core set of PROX1-regulated We thus compared the proliferation rates between U-343 genes, we filtered genes that were correlated with PROX1 in MG-control and U-343 MG-PROX1, and between U-343 TCGA, CCLE, and HGCC resource (Fig. 4C; ref. 27). In an MGa-control and U-343 MGa-shPROX1 cell lines by cell count- extended exploration of these datasets, we found similarities ing. Overexpression of PROX1 increased proliferation of U-343 between gene expression patterns in glioblastoma tumor mater- MG-PROX1 cultures (Fig. 5B), whereas PROX1 suppression ial and cell cultures regarding PROX1 correlated genes, and decreased the proliferation rate of U-343 MGa (Fig. 5C). In the identified GO terms suggesting involvement of PROX1 in several RNA-seq experiment, we noticed changes in the levels of several biological processes including regulation of nervous system devel- cyclin and cyclin-dependent kinase (CDK) genes following opment and chromatin modifications (Supplementary Fig. S2). PROX1 modulation (Fig. 5D). To investigate if the altered The analysis revealed 82 intersecting PROX1 correlated genes proliferation was paralleled by changes in levels of cell-cycle between CCLE, HGCC, and TCGA (Fig. 4C). Strikingly, of these regulatory proteins, we performed immunoblotting analysis of the top 10 PROX1-upregulated genes, ranging from a 203- to a cyclins. We found increased levels of cyclins A1 (CCNA1), B1 15-fold increase, all have been reported to be involved in neuro- (CCNB1), D1 (CCND1), and E1 (CCNE1) proteins in U-343 glial development, including SEMA6A, NES, PTPRZ1, FABP7, MG-PROX1. PROX1 suppression resulted in decreased

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Figure 4. PROX1 regulation of glioblastoma expression-based subtypes. A, Changes of subtype defining genes upon PROX1 overexpression in U-343 MG compared with control assessed by RNA-seq. Genes in the pink or blue areas are regulated in the corresponding direction supporting the subtype change and add to a positive index score. Index value of 100 is full negative change of subtype, 0 is neutral, and 100 is a full positive subtype change. Gray, genes with no available measurement. PN, proneural; CL, classical; MES, mesenchymal; N, neural. B, The same illustration as for A, but corresponding to suppression of PROX1 in U-343 MGa versus control. C, Venn diagram representing number of genes positively regulated by PROX1 more than two-fold according to the RNA-seq data and found to have positive correlation with PROX1 in TCGA clinical data (r > 0.3). Highlighted are the genes with the highest increased expression levels by PROX1 overexpression (top 10). D, Bar graph depicting regulation of stemness signature genes from Patel and colleagues (4). by PROX1 overexpression in our in vitro system. E, Heatmaps representing gene expression of top 100 correlated genes with a PROX1 proxy profile in single-cell analysis data from five glioblastomas. The cells are arranged (from high to low) according to their correlation to the proxy profile in each tumor. Red, high gene expression; blue, low expression. Bar graphs represent PROX1 regulation of these genes based on the RNA-seq analysis of PROX1 overexpression in U-343 MG.

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Figure 5. PROX1 overexpression increased cell proliferation, coinciding with changes of cell-cycle regulators. A, Boxplot data extracted from HGCC showing higher PROX1 expression in glioblastoma cultures with tumor-initiating capacity assessed by intracranial tumor formation (red) according to ref. 27. B, Increased rate of cell proliferation in U-343 MG-PROX1 as compared with U-343 MG parental line, determined by cell counting at the days indicated. C, Bar graphs showing decreased cell proliferation rate upon PROX1 suppression in U-343 MGa compared with control. D, Heatmap illustration of RNA-seq experiment data indicating changes of cyclins and CDK gene expression following PROX1 modulation. Red, high expression; blue, low expression. E, Immunoblots showing levels of cyclin A1, cyclin B1, cyclin D1, cyclin E1, p21, p27, p57, and p53 in U-343 MG upon PROX1 overexpression and upon suppression in U-343 MGa cells. Immunoblot of b-actin protein was used as loading controls.

cyclins A1 and E1 in U-343 MGa-shPROX1, whereas no notice- PROX1 interacts directly and colocalizes with THRAP3 in the able change was observed for cyclin D1 or cyclin B1 (Fig. 5E). In nucleus addition, we observed decreased p53 (TP53) and increased The function of PROX1 and its regulation remains poorly p21 (CDKN1A), p27 (CDKN1B), and p57 (CDKN1C) protein understood in molecular details. We thus performed experiments levels after PROX1 overexpression, whereas the levels to identify proteins that interact with PROX1. Initially, we used an remained unchanged in U-343 MGa-shPROX1 compared with osteosarcoma cell line that stably expresses PROX1-FLAG or the control (Fig. 5E). In summary, we found increased or empty-FLAG. A nuclear complex co-IP, followed by mass spec- decreased proliferation upon PROX1 overexpression or suppres- trometry analysis, identiﬁed proteins associated with PROX1, of sion, respectively, which coincided with changes of cell-cycle which none has previously been described as a PROX1 interacting regulators. protein (Supplementary Fig. S3). One of the identiﬁed PROX1

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associated proteins was THRAP3 (thyroid hormone receptor and protein levels (Fig. 7C and D), and SOX2 suppression in associated protein 3, aka TRAP150). THRAP3 peptides detected U-343 MGa decreased PROX1 protein levels (Fig. 7E). However, by MS/MS sequencing are shown (Fig. 6A). Subsequently, we suppression of PROX1 in U-343 MGa did not decrease SOX2 confirmed the interaction of endogenous PROX1 with THRAP3 protein levels (Fig. 7D). Given that CDK2 activity can stabilize protein in the U-343 MGa cells via a reciprocal co-IP experiment SOX2 (38), we investigated if treatment of U-343 MGa cells with (Fig. 6B). Given the important role of THRAP3 in several cellular the CDK2 inhibitor CVT-313 compound would result in processes related to transcriptional activities, we decided to inves- decreased PROX1 protein levels and found that CVT-313 treat- tigate a functional link between THRAP3 and PROX1. We found ment decreased both SOX2 and PROX1 protein levels within that both proteins localize to the nucleus (Fig. 6C). Upon THRAP3 24 hours (Fig. 7F). It was confirmed that treatment of cells with suppression following 5 days of siRNA treatment PROX1 levels 10 mmol/L of the compound for 24 hours also decreased cyclin E1 were found to have increased (Fig. 6D). Similarly, 2-day THRAP3 levels (Fig. 7F). Together, these data indicate that SOX2 controls siRNA treatment also led to increased PROX1 protein levels in PROX1 expression and that CDK2 inhibition leads to decreased U-343 MGa as well as in U-343 MG-PROX1 cells (Fig. 6E). In line levels of both SOX2, PROX1, and cyclin E1. It was recently with the result that PROX1 increased GFAP transcript and protein reported that THRAP3 binds to SOX9 and regulates transcription levels (Fig. 2F), we found that GFAP protein levels also increased (39). Also, we have previously reported a subset of glioblastoma upon THRAP3 suppression, although seemingly independent of cell cultures with high expression of SOX2 and SOX9 (22). We PROX1 (Fig. 6F). To investigate if the increase in PROX1 and therefore investigated the correlation of expression between GFAP levels was due to transcriptional effects, we also assessed SOX2, SOX9, and THRAP3 in glioblastoma samples and found transcript levels and found an increase of PROX1 and GFAP that THRAP3 was highly correlated with SOX2 and SOX9 (Fig. 7G expression upon THRAP3 suppression (Fig. 6G). In parallel, no and H). Including the results presented in Fig. 6i, this connects transcript changes were observed for the oligodendroglial and the expression levels of SOX2, THRAP3, PROX1, and SOX9 in neuronal makers OLIG2 and MAP2, respectively (Fig. 6G). To glioblastoma samples. investigate the association between THRAP3 and PROX1 tran- THRAP3 scripts in glioma, we explored the TCGA data. expression Discussion was significantly less in glioma grade IV as compared with grade II (P < 0.0001) and grade III (P < 0.0001) tumors (Fig. 6H), Results presented in this work indicate that PROX1 mRNA level displaying a similar pattern to PROX1 expression in the same fluctuates with tumor grade of gliomas, and is expressed at a lower samples (cf. Fig. 1A). Moreover, a positive correlation was found level in grade IV gliomas. In particular, for grade IV gliomas we between THRAP3 and PROX1 transcript levels in glioblastoma found that the mesenchymal subtype was associated with rela- (Pearson r ¼ 0.58; Fig. 6I). Finally, patients with high or low tively low PROX1 levels. In the light of recent findings about THRAP3 transcript levels differed with regard to overall and tumor heterogeneity and the intermixture of mesenchymal and disease-free survival (Fig. 6J, K, and L). A survey of genes with nonmesenchymal gene expression subtype cells (4), the data the highest correlation to both PROX1 and THRAP3 expression in present a mechanism of how these subtypes may be regulated glioblastoma samples revealed overlaps with hallmark gene sets by PROX1 during tumor cell evolution. In this analysis PROX1 is including oxidative phosphorylation, biological processes less expressed in the mesenchymal-like U-343 MG cell line as including nucleoside triphosphate metabolic process, and cellular compared with its sibling line U-343 MGa, and the clonal deri- compartments such as respiratory chain and mitochondria (Sup- vatives thereof, U-343 MGa Cl2:6 and U-343 MGa 31L. According plementary Fig. S3). Taken together, these results identify to a suggested model of tumor progression, from a nonmesench- THRAP3 as a novel interacting partner for PROX1, and suggest ymal to a mesenchymal subtype (40), the U-343 MG cells should its involvement in the regulation of PROX1 and GFAP transcript then have arisen from a U-343 MGa ancestry clone where PROX1 and protein levels. expression has been lost. Per our gene expression analysis, the U-343 MGa culture became more mesenchymal-like upon PROX1 Correlation and regulatory connection of PROX1 and SOX2 suppression, and conversely U-343 MG shifted to a nonmesench- in glioblastoma ymal subtype profile upon PROX1 overexpression. Thus, our data SOX genes are key regulators of embryonic development of the show that PROX1 can regulate the glioblastoma subtype in a CNS, and its maintenance in adults (36). Specifically, SOX2 has bi-directional manner. In extension, the switch between glio- been reported to bind to the PROX1 promoter in a global ChIP- blastoma transcriptional subtypes is reversible and thus allows seq analysis (37), which suggests that SOX2 could be a determi- glioblastoma cells to fluctuate between subtypes and give rise to nant for PROX1 regulation. It was recently shown that SOX2 is heterogeneous tumor cell populations. The subtype gene stabilized through phosphorylation by cyclin E-CDK2 complexes expression signature is relatively stable for the U-343 cultures in glioma (38). Given that PROX1 was found to affect expression in vitro, suggesting an underlying genetic or epigenetic cause. of cyclins in glioma cells, we investigated the connection between Moreover, whether external queues from neighboring cancer PROX1, SOX2, and cyclins. As illustrated in Fig. 2A, we noted a cells and tumor microenvironment also may affect PROX1 positive correlation between PROX1 and SOX2 protein levels in a levels remains to be studied. Beyond strictly clonal differences, panel of glioma cell lines. In addition, we found a significant maintained by genetic or epigenetic events, this could be an positive correlation between expression of PROX1 and SOX2 in underlying explanation for previous observations where not 375 cases from TCGA clinical data (r ¼ 0.43; Fig. 7A), in 48 all cancer cells within single tumors displayed similar PROX1 glioblastoma cell cultures from HGCC (r ¼ 0.38), and in 45 protein levels (14). Therefore, our findings underscore glioblastoma cell lines from CCLE (r ¼ 0.55; Fig. 7B). In line with PROX1 function in gliomas where its decreased mRNA level SOX2 being an upstream regulator of PROX1 we found that SOX2 is connected with tumor cell evolution and intratumoral overexpression in U-343 MG caused elevated PROX1 transcript heterogeneity.

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PROX1 is not an established oncogene or tumor suppressor, dent experimental settings. IHC analysis by Elsir and colleagues þ þ þ but results presented here place it downstream of SOX2 that is a identified up to 80% PROX1 /MAP2 and around 30% PROX1 / þ þ þ prominent glioblastoma cancer driver due to its focal amplifica- GFAP , but only 1% to 2% MAP2 /GFAP cells in high-grade tion and tumor-initiating capacity (41, 42). The high correlation gliomas, indicating that tumor cells represent either neuronal or of PROX1 and SOX2 expression in tissue, and previous reports of glial differentiation pathways in vivo (14). Furthermore, Prox1 SOX2 binding to the PROX1 promoter identified by ChIP-seq depletion has been shown to reduce the number of stem cells and analysis of glioblastoma (37) highly suggest that PROX1 is reg- the rate of cell proliferation in intestinal tumor growth (13). ulated by SOX2. Of note, our GSEA of glioblastoma cell cultures According to the present data analysis of glioblastoma samples, from CCLE with high or low PROX1 expression identified a PROX1 expression correlates with GFAP and is frequent in the significant enrichment of SOX2 target genes. Thus, in addition proneural and classical subtypes. Based on these observations, we to PROX1 being part of a normal CNS developmental program, speculate that PROX1 marks a transitory stage in the evolution of we propose that abnormally regulated PROX1 is an important gliomas, possibly coinciding with the choice between neuronal component of astrocytic tumor formation and growth. The and glial cell developmental paths. Similarly, studies on the role decreased proliferation rate observed upon PROX1 suppression of Prox1 in mouse CNS development indicate a crucial function at in U-343 MGa raises the question if PROX1 has a growth pro- this stage of CNS development (10, 43). moting effect in early stage tumors; whether it decreases with PROX1 is part of several complexes involved in development increasing grade and potentially regulates transcriptional sub- and tumorigenesis including NCoR and HDAC3 (44), as well as types during tumor progression. It has been reported that the PGC-1a and ERRa, which together with BMAL1 play central roles tumor-initiating capacity in stem-like glioma cell cultures with in the transcriptional control of energy homeostasis and biolog- high growth rate, as opposed to mesenchymal-like cultures with ical clocks in the mice liver (45, 46). Here we identified THRAP3 low proliferative rate and tumor-initiating capacity, is maintained as a novel binding partner for ectopic and endogenous PROX1 by SOX2 (22). Consistent with this, in our analysis of HGCC data in U2-OS and U-343 MGa cells, respectively. Suppression of (27), PROX1 expression was also associated with higher tumor THRAP3 in the glioblastoma culture U-343 MGa resulted in initiating capacity. However, we found that glioblastomas with increased PROX1 and GFAP protein levels, findings that physi- lower PROX1 levels, mesenchymal subtype, have worse outcome. cally and functionally connects PROX1 and THRAP3. Moreover, Similarly, Patel and colleagues demonstrated that glioblastomas THRAP3 suppression increased PROX1 and GFAP transcripts. with a high representation of proneural cells were associated with Interestingly, Sono and colleagues recently demonstrated longer survival, compared with those with a high intermixture of THRAP3 as an interactor with SOX9, where the two proteins mesenchymal cells (4). Furthermore, the glioblastoma cancer together inhibited transcriptional activity during chondrogenesis stem cell signature was found strongest in individual cells con- (39). In the analysis of glioma we found high correlation between forming to the proneural and classical subtypes, but underrepre- PROX1, THRAP3, SOX2, and SOX9. In addition to PROX1, SOX2 sented in cells of the mesenchymal subtype. This presents a binds to the promoter region of THRAP3 and SOX9 (37). Togeth- conceptual contradiction, where tumor data connects the mes- er, these results imply that SOX2 acts upstream of transcriptional enchymal subtype with poor outcome, whereas in an experimen- complexes composed of PROX1 or SOX9, and that THRAP3 is a tal setting nonmesenchymal stem-like cells display higher tumor- modulator of their transcriptional activity. Further, THRAP3 is igenic potential. We speculate that as gliomas become more known as a clock-regulated component of CLOCK–BMAL1 com- mesenchymal when they progress, mesenchymal subtype plexes that promotes CLOCK–BMAL1 transcriptional activity and glioblastomas may represent more progressed tumors that per se is important for circadian clock function (47). In the GSEA, we have shorter survival. Many experimental models do not reflect identified low PROX1 levels to be associated with increased this and thus may be the underlying cause for this discordance. levels of ERRa target genes, described in ref. 48. Based on our Furthermore, the intermixture of nonmesenchymal and mesen- and others' results this ties PROX1, through THRAP3, to ERRa chymal cells may have tumorigenic effects that are not observed in and BMAL1. We thus speculate that THRAP3 interacts with tumor models with low clonal heterogeneity. PROX1 to modulate PROX1 transcriptional function and may The present results show that PROX1 and GFAP are positively take part in regulation of developmental pathways and of the correlated in glioblastomas and cell lines datasets, and GFAP cell-cycle machinery by direct or indirect control of metabolic protein is decreased upon PROX1 suppression in two indepen- pathways, for example via changes of estrogen signaling (49).

Figure 6. PROX1 interacts directly with THRAP3 in the nucleus and is upregulated following THRAP3 suppression. A, THRAP3 peptides detected by MS-MS sequencing, following a nuclear complex co-IP. B, Double nuclear complex co-IP experiment confirming interaction of endogenous PROX1 with THRAP3 protein using nuclear fraction from U-343 MGa glioma cells. Immunoprecipitation was conducted with a rabbit antibody against THRAP3, and the immunoprecipitate and input fractions were blotted with a goat PROX1 antibody. Shown is also a THRAP3 immunoblot (on the left). No band was detected in lanes corresponding to the negative control (no antibody), nor in IgG control lanes. Shown on the right side are immunoprecipitates with two different antibodies against PROX1 (Ab1 and Ab2) and the input fraction blotted with THRAP3 antibody. THRAP3 was found present in PROX1 immunoprecipitates captured by either antibody and more efficiently with Ab 1. Also shown is an immunoblot of PROX1. C, Colocalization of PROX1 and THRAP3 in the nucleus. D, Immunoblots showing increased PROX1 levels following THRAP3 suppression evaluated after 120 hours of siRNA treatment. E, Immunoblots showing PROX1 levels following THRAP3 suppression evaluated after 48 hours of siRNA treatment. F, Immunoblots showing GFAP protein levels following THRAP3 suppression evaluated after 48 hours of siRNA treatment. G, qRT-PCR analysis in U-343 MGa upon 48 hours of siTHRAP3 treatment as compared to control. H, Comparison of THRAP3 expression in glioma grades II to IV. I, THRAP3 correlation with PROX1 in TCGA glioblastoma data extracted from (http://cbioportal.org). J, Analysis of THRAP3 expression z-scores cutoffs in TCGA data for 206 patients with glioblastoma (http://cbioportal.org). z-score 0.3 yielded significant results for differential survival. K and L, Kaplan–Meier curves using z-score cutoff 0.3 for THRAP3 for disease-free and overall survivals.

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Figure 7. Correlation and regulatory connection of PROX1 and SOX2 in glioblastoma. A, Scatterplot for coexpression of PROX1 and SOX2 (r ¼ 0.43) in TCGA glioblastoma clinical data extracted from http://cbioportal.org. B, Scatterplots depicting expression of PROX1 and SOX2 in 45 glioblastoma lines from CCLE (r ¼ 0.55) and in 48 cultures from HGCC (r ¼ 0.3827). C, qRT-PCR analysis indicating induction of PROX1 mRNA expression in a U-343 MG cell line with stable overexpression of SOX2, as compared to the control. D, Immunoblots indicating overexpression of PROX1 protein in U-343 MG-SOX2. E, Immunoblots indicating decreased PROX1 levels following 48 hours of siSOX2 treatment of U-343 MGa cells. F, Treatment of U-343MGa cells with CDK2 inhibitor CVT-313 (10 mmol/L) for 24 hours resulted in decreased PROX1, SOX2, and cyclin E1 levels, assesses by immunoblotting. Shown is also an immunoblot of CDK2. G and H, Correlation analysis for THRAP3 expression levels versus SOX2 and SOX9 in TCGA glioblastoma samples.

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PROX1 has previously been shown to control CCNE1 by direct yield heterogeneous cell populations. Finally, CDK2-inhibitors binding to its promoter (50). Adding to this, we show that PROX1 may pose an opportunity to target cell growth of glioblastoma affects cyclin protein levels and growth rate in glioblastoma. cells with high PROX1 expression. Berger and colleagues unraveled a critical function for CCNE1 during neural cell fate specification in Drosophila, through inter- Disclosure of Potential Conflicts of Interest fering with Prospero localization to the nucleus and thereby No potential conflicts of interest were disclosed. regulating its function (50, 51). Based on mutant analysis in the CNS of flies, it has been proposed that cyclin E function in Authors' Contributions specifying a neuronal as opposed to glial cell fate is independent Conception and design: K.M. Goudarzi, J. Bartek, M. Nister, M.S. Lindstrom,€ D. H€agerstrand of its established role in G1–S phase transition (51). Furthermore, Development of methodology: K.M. Goudarzi CCNE1/CDK2 has recently been shown to stabilize pluripotency Acquisition of data (provided animals, acquired and managed patients, factors, on a protein level, including SOX2, NANOG, and provided facilities, etc.): K.M. Goudarzi, M. Guo, M. Nister, M.S. Lindstrom€ POU5F1 (OCT4; ref. 38). These observations together with data Analysis and interpretation of data (e.g., statistical analysis, biostatistics, presented here suggest a feed forward loop where PROX1 computational analysis): K.M. Goudarzi, J.A. Espinoza, D. H€agerstrand enhances CCNE1 transcription, CCNE1 in complex with CDK2 Writing, review, and/or revision of the manuscript: K.M. Goudarzi, € € stabilizes SOX2, and finally SOX2 enhances PROX1 transcription. J.A. Espinoza, J. Bartek, M. Nister, M.S. Lindstrom, D. Hagerstrand Administrative, technical, or material support (i.e., reporting or organizing This places PROX1 as a link between cell proliferation and data, constructing databases): M. Guo stemness regulation in glioblastoma. The factors that might Study supervision: M. Nister, M.S. Lindstrom,€ D. H€agerstrand govern such a loop remain to be studied, but one could speculate that the regulation of such a feed forward loop is out of control in Acknowledgments glioblastoma. The authors appreciate discussions with the members of the Nister labora- The characteristic intratumoral heterogeneity of glioblastoma is tory, as well as the Bartek group and the SciLifeLab research community who suggested to reflect neural development and is likely a key to helped make this article possible. This work was supported by KID-funding € understanding treatment failure (4). Based on the results in this (to K.M. Goudarzi, 3595/2012), Åke Wiberg stiftelse (to M.S. Lindstrom, 390316483), Karolinska Institutet (to M.S. Lindstrom,€ 1885/12-226 and to paper PROX1 can be decreased by the CDK inhibitor CVT-313 and D. H€agerstrand, 2014fobi41302), Magnus Bergvall's stiftelse (to M.S. Lindstrom€ its SOX2 protein destabilizing effect (38). This places CVT-313 as a and to D. H€agerstrand, 2014-00600), King Gustaf V's Jubilee Foundation (to M. growth inhibitory drug that would target glioblastomas with high S. Lindstrom,€ 164102), and the Swedish Research Council (to M.S. Lindstrom,€ PROX1 expression. In contrast, a CVT-313 induced decrease K2012-99X-21969–01–3). M. Guo was supported by the Chinese Scholarship of PROX1 would be predicted to transition cells from a non- Council. M. Nister was supported by grants from the Swedish Cancer Society mesenchymal to a mesenchymal subtype and thus potentially (CAN 2014/836, contract 160334; CAN 2017/737, contract 170659), the Cancer Society in Stockholm (2015-151213), the Swedish Research Council promote tumor progression. Further experiments are required to (K2014-67X-15399-10-4), the Swedish Childhood Cancer Foundation understand how the balance between the growth inhibitory and (PR2014-0021; 2017-0002), Karolinska Institutet (2016fobi47658), and the transition regulation of PROX1 translates in a tumor setting. In Stockholm County Council (SLL), and J. Bartek by grants from the extension, the benefit of transitioning cells from a nonmesench- Swedish Cancer Society (contract 170176) and the Swedish Research Council ymal to a mesenchymal subtype or vice versa as a therapeutic (K2014-46602-117891-30). approach remains to be studied. In summary, this work highlights the utility of PROX1 as a The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked prognostic marker and adds biological insight to its role in advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate glioblastoma, where it maintains a neural stemness gene expres- this fact. sion profile and a proliferative state with high levels of G1-cyclins. Furthermore, loss of PROX1 transitions proneural cells into a Received April 25, 2018; revised July 1, 2018; accepted August 16, 2018; mesenchymal subtype, which during tumor evolution would published first August 22, 2018.

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5916 Cancer Res; 78(20) October 15, 2018 Cancer Research

Reduced Expression of PROX1 Transitions Glioblastoma Cells into a Mesenchymal Gene Expression Subtype

Kaveh M. Goudarzi, Jaime A. Espinoza, Min Guo, et al.

Cancer Res 2018;78:5901-5916. Published OnlineFirst August 22, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-18-0320

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