PBX3 Is Part of an EMT Regulatory Network and Indicates Poor

Published OnlineFirst February 1, 2018; DOI: 10.1158/1078-0432.CCR-17-2572

Biology of Human Tumors Clinical Cancer Research PBX3 Is Part of an EMT Regulatory Network and Indicates Poor Outcome in Colorectal Cancer Sebastian Lamprecht1, Markus Kaller2, Eva Marina Schmidt1, Cristina Blaj1, Tobias S. Schiergens3, Jutta Engel4, Andreas Jung1,5,6, Heiko Hermeking2,5,6, Thomas G.P. Grunewald€ 1,5,6,7, Thomas Kirchner1,5,6, and David Horst1,5,6

Abstract

Purpose: Colorectal cancers are composed of phenotypically sion was induced by WNT activation and by the EMT transcription different tumor cell subpopulations within the same core genetic factors SNAIL and ZEB1, whereas these effects were mediated background. Here, we identify high expression of the TALE indirectly through microRNA miR-200. PBX3 was required for a transcription factor PBX3 in tumor cells undergoing epithelial– full EMT phenotype in colon cancer cells. On the protein level, mesenchymal transition (EMT), analyze PBX3 regulation, and PBX3 expression indicated poor cancer-speciﬁc and disease-free determine clinical associations in colorectal cancer. survival in a cohort of 244 UICC stage II colorectal cancers, and Experimental design: We used transcriptomic and in situ was associated with metastasis in a case–control collection con- analyses to identify PBX3 expression in colorectal cancer and cell sisting of 90 cases with or without distant metastasis. On the biology approaches to determine its regulation and function. mRNA level, high PBX3 expression was strongly linked to poor Clinical associations were analyzed in independent tissue collec- disease-free survival. tions and gene expression datasets of colorectal cancers with Conclusion: PBX3 is a novel indicator of EMT in colorectal recorded follow-up data. cancer, part of an EMT regulatory network, and a promising Results: PBX3 was expressed in tumor cells with high WNT prognostic predictor that may aid in therapeutic decision making activity undergoing EMT at the leading tumor edge of colorectal for patients with colorectal cancer. Clin Cancer Res; 1–13. 2018 cancers, whereas stromal cells were PBX3 negative. PBX3 expres- AACR.

Introduction Colon cancer cells with high WNT activity have especially been associated with a specific phenotype. These tumor cells are Colorectal cancer ranks third in cancer incidence among men typically located at the leading tumor edge, show a less differ- and women and is a major cause of cancer morbidity and mortality entiated morphology, and infiltrate surrounding stromal tissue (1). More than 80% of colorectal cancers have driver mutations in (4). Moreover, they are characterized by loss of epithelial APC or b-catenin that over activate WNT signaling in these tumors differentiation with reduced expression of the cell adhesion (2). Although these mutations are present in all clonally derived molecule E-cadherin, and increased expression of mesenchy- colon cancer cells, WNT signaling still remains regulated in these mal markers, including vimentin, fibronectin, and LAMC2 tumors, which is reflected by coexistence of tumor cell subpopu- (5, 6). This phenotype is termed epithelial–mesenchymal transi- lations with relatively low or high WNT activity (3). tion (EMT) and is regulated by transcription factors of the ZEB and SNAIL families, including ZEB1, which is induced by WNT signaling in colon cancer (7). Downstream effects of ZEB1 1Pathologisches Institut, Ludwig-Maximilians-Universitat€ Munchen,€ Germany. 2Experimental and Molecular Pathology, Pathologisches Institut, Ludwig-Max- then are either transduced through direct transcriptional acti- imilians-Universitat€ Munchen,€ Germany. 3Department of General, Visceral, vation, or indirectly through repression of microRNAs of the Transplantation, Vascular and Thoracic Surgery, Ludwig-Maximilian-Universitat€ miR-200 family that target EMT-related genes (8). Because of Munchen,€ Germany. 4Munich Cancer Registry (MCR), Department of Medical the promotion of tumor infiltration by EMT, high WNT activity Information Processing, Biometry and Epidemiology (IBE), Ludwig-Maximilians- therefore is a crucial driver of colon cancer invasion and € € 5 Universitat Munchen, Germany. German Cancer Consortium (DKTK), Heidel- progression. Identifying factors that are involved in EMT regu- berg, Germany. 6German Cancer Research Center (DKFZ), Heidelberg, Germany. lation through WNT, thus, may hold keys for a better under- 7Max-Eder Research Group for Pediatric Sarcoma Biology, Pathologisches Institut, Ludwig-Maximilians-Universitat€ Munchen,€ Germany. standing of the malignant biology of colorectal cancer. Pre–B-cell leukemia homeobox transcription factor 3 (PBX3) Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). belongs to a family of evolutionary conserved three-amino- acid-loop-extension (TALE) homeodomain transcription fac- S. Lamprecht and M. Kaller contributed equally to the article. tors. These are known to serve as cofactors for homeobox Corresponding Author: David Horst, Ludwig-Maximilians-Universitat€ Munchen,€ (HOX) proteins and are physiologically involved in regulation € Thalkirchner Straße 36, 80337 Munchen, Germany. Phone: 49-89-2180-73611; of gene expression during embryonic development (9, 10). In Fax: 49-89-2180-73727; E-mail: [email protected] cancer, PBX3 has been functionally linked to the development doi: 10.1158/1078-0432.CCR-17-2572 of certain forms of leukemia (11). Furthermore, PBX3 has been 2018 American Association for Cancer Research. associated with tumor progression and metastasis in gastric and

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clinical endpoint, documented in 76 patients (31.1%) either as Translational Relevance tumor recurrence or metastasis. Survival data were censored High WNT activity and epithelial–mesenchymal transition when case follow-up was discontinued, or when patients had (EMT) are drivers and hallmarks of colon cancer progression. died of reasons other than colorectal cancer. Variables consid- Here, we identify PBX3 as a new indicator of EMT and part of ered for inclusion models were age, sex, T-stage, tumor grade, an EMT regulatory network that is associated with poor prog- and neo-/adjuvant therapy. At a power of 0.8 with a¼0.05, nosis in colon cancer. Assessment of PBX3 in colon cancer relative risks of 2.2 for cancer-specific survival and 1.9 for tissues may improve risk stratification for patients with colon disease-free survival could be detected in this collection cancer through gauging EMT, which is not yet adequately (19), indicating sufficient sample size for our study design. reflected by other routinely assessed clinical parameters. For the metastasis collection, we chose a case–control design that included tumor specimens of 90 patients who were surgically resected or biopsied at the LMU between 1994 and 2005. None of them were part of the survival collection. Half of the patients had colon cancer (12–14).Ingastriccancer,PBX3hasbeenshown colon cancers with synchronous liver metastasis (UICC stage IV), to induce an infiltrative tumor cell phenotype with upregula- diagnosed by clinical imaging or liver biopsy. Controls consisted tion of vimentin and repression of E-cadherin (15). In addition, of patients with colon cancer without distant metastasis at the – microRNAs that were shown to regulate PBX3 included mem- time of diagnosis (UICC stages I III) and with disease-free sur- bers of the miR-200 family (16). These data suggested onco- vival of at least 5 years after primary surgical resection. Cases and genic features of PBX3 in colorectal and other cancers, and controls were matched by tumor grade (according to WHO 2010), provided a first link between PBX3 and EMT. However, the T-category, and tumor location (all tumors were right-sided colon regulation of PBX3 in colon cancer and its contribution to cancers), resulting in 45 matched pairs. Sample size was based on tumor progression still are incompletely understood. previous experience and limited by the availability of tumor Here, we aimed to identify transcription factors that are linked material. to tumor cell heterogeneity and differential WNT signaling activity Immunohistochemistry and immunofluorescence in colorectal cancer. In this context, we identified PBX3 and Immunohistochemistry was done on 5-mm tissue sections on a subsequently determined its regulation in colon cancer in detail. Ventana Benchmark XT autostainer with ultraView Universal DAB We found that PBX3 expression is regulated by WNT signaling detection kits (Ventana Medical Systems) using the antibodies and EMT, and that it is required for a full EMT phenotype in and concentrations listed in Supplementary Table S1. Expression colon cancer cells. Furthermore, we evaluated its potential as a of PBX3 was evaluated blinded from clinical outcome by aver- prognostic biomarker for patients with colorectal cancer. aging the percentage of PBX3-stained tumor cells in all TMA cores of a tumor. To determine intratumoral co-localization of PBX3 Materials and Methods and b-catenin or LAMC2, we measured staining intensities in Clinical cases tumor cells of different areas, including the leading tumor edge Study design was based on the REMARK criteria (17). For- and the tumor center, on serial tumor sections using ImageJ malin-fixed, paraffin-embedded (FFPE) specimens were (20). For immunofluorescence, cultured cells were fixed in 4% obtained from the archives of the institute of pathology of the paraformaldehyde for 10 minutes, permeabilized with 0.2% Ludwig-Maximilians-University Munich (LMU). A frozen sam- TritonX100 for 15 minutes and blocked with 3% BSA in PBS for ple of normal mucosa and corresponding colon cancer tissue 30 minutes. Cells then were incubated sequentially at room was provided by the biobank under administration of the temperature with primary antibodies listed in Supplementary foundation Human Tissue and Cell Research (HTCR; ref. 18). Table S1 and then with Alexa Fluor 488- or 568-labeled secondary For patients with cancer, follow-up data were recorded by the antibodies (Abcam). F-actin was visualized with Alexa Fluor Munich Cancer Registry. Specimens were anonymized, and the 647-labeled Phalloidin (Invitrogen). Nuclei were counterstained need for consent was waived by the institutional ethics com- with DAPI (Vector Laboratories, 1:500). Confocal fluorescence mittee of the Medical Faculty of the LMU. Tissue microarrays of images were taken on an LSM 700 laser scanning microscope FFPE cancer specimens (TMAs) were generated with six repre- using the ZEN software (Carl Zeiss). sentative 1-mm cores, including tumor edges and tumor centers of each case, using hematoxylin and eosin–stained tissue sec- Gene-expression datasets and GSEA tions as templates. Individual samples of normal colonic Gene-expression datasets were retrieved from the Gene mucosa also were included. Expression Omnibus (21). For comparative analyses of colon For the survival collection, we included 260 randomly select- cancer cell subpopulations with low and high WNT activity, ed patients of a cohort of 949 patients diagnosed with UICC datasets GSE32408 and GSE17375 were used (3, 22). Micro- stage II colorectal cancer at the LMU between 1994 and 2007 array data were normalized simultaneously with Robust Multi- that underwent surgical resection. Sixteen cases dropped out array Average (RMA; ref. 23) using custom brainarray CDF (v19, due to lack of sufficient tumor material, resulting in a final ENTREZG; ref. 24) in R (www.r-project.org), which yielded one collection of 244 patients with a median follow-up time of optimized probeset per gene, as previously described (25, 26). 4.9 years. Forty-three patients (17.6%) had received adjuvant or Data then were filtered for transcription factors based on anno- neoadjuvant radiation and/or chemotherapy. For tumor-spe- tations by the human protein atlas (27). For the analysis of cific survival, colorectal cancer attributed death was defined as colon cancer samples, we retrieved datasets GSE14333 and clinical endpoint, and was documented in 52 patients (21.3%). GSE39582, based on the availability of matched transcriptome For analysis of disease-free survival, tumor progression was the and clinical data, and normalized them simultaneously in the

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same manner as described previously. Pearson correlations of FuGENE 6 (Promega). Firefly luciferase activity was measured PBX3 expression and expression of all other genes represented with dual-luciferase Reporter Assays (Promega) after 24 hours within these datasets then were calculated, and genes were and normalized to Renilla luciferase activity. Luminescence was ranked accordingly. GSEA analyses (28) then were done using measured with an Orion II luminometer (Berthold). this ranked gene list against curated sets of EMT core signatures (29, 30). Heatmaps for selected genes were generated with Immunoblotting GENE-E (software.broadinstitute.org/GENE-E/). Immunoblotting was done using whole-cell lysates supplemented with protease and phosphatase inhibitors (Roche), as Genetic vectors previously described (35). Proteins were separated by SDS-PAGE, The DOX inducible episomal pRTR vector system and transferred onto polyvinylidene difluoride membranes (Merck pRTR-SNAIL-VSV were described previously (31). For Millipore) and incubated with primary antibodies listed in Sup- pRTR-ZEB1-VSV, we excised the ZEB1 coding sequence from plementary Table S1. Bands were visualized using horseradish pcDNA4hismaxCZEB1, a gift from Janet Mertz (32), and peroxidase (HRP)-conjugated secondary mouse (Promega) or inserted it into SfiI restriction sites of pRTR. For microRNA- rabbit (Sigma) antibodies and chemiluminescent HRP Substrate 0 binding luciferase reporter constructs, we amplified the 3 -UTR (Millipore). Bands then were quantified using ImageJ (20) and of the human PBX3 gene containing the miR-200–binding site normalized to a-tubulin or b-actin. by PCR, using primers 50-GATCAGAGACTGGTAGCATCG-30 0 0 and 5 -AATCATGAAAGCAAAAAGTTTATTC-3 ,andcDNAfrom Real-time quantitative PCR SW480 cells as template, and inserted it into pGL3 (Promega). Total RNA was isolated using QIAzol Lysis Reagent (Qiagen) To insert mutations into the miR-200 seed-matching sequence, and cDNA was generated from 500 ng total RNA per sample using we then applied site directed mutagenesis using the Quik- QuantiTect Reverse Transcription (Qiagen). Real-time quantita- Change Mutagenesis Kit according to the manufacturer's pro- tive PCR (qRT-PCR) then was done using Fast SYBR Green Mix tocol (Agilent). For analysis of WNT/b-catenin activity within (Applied Biosystems) on a LightCycler 480 (Roche), applying the PBX3 promoter, 2.5 kbp upstream of the PBX3 transcription 40 cycles of amplification at 95C (1 sec), 60C (20 sec), and 72C start site containing TCF4-binding sites were amplified from (1 sec). Primers are listed in Supplementary Table S2. qRT-PCR human BAC clone CTD-2309J17 (CalTech BAC library) using results were first normalized to GAPDH mRNA levels in the Pfu Polymerase (Thermo Scientific) and primers 5'-CTCTA- same sample and then to levels of the corresponding transcript 0 0 AGCGCTTTGCGATTG-3 and 5 -AGCATCCTGGATTGATCGT- in control-treated tumor cells. C-30. PCR products then were inserted into pBV-Luc vector (a gift from Bert Vogelstein, Addgene plasmid #16539). Synthetic Statistical analyses DNA sequences (IDT) then were used to replace TCF4-binding Survival was analyzed by the Kaplan–Meier method and fi fi sites by mutated sites. Modi ed vector elements were veri ed groups were compared with the log-rank test. Optimal cutoffs by restriction analysis and sequencing. for continuous variables were selected by receiver operating characteristic (ROC) curve analyses and Youden's index. Cox Cell culture, transfections, and luciferase assays proportional hazards model was used for uni- and multivariate SW1222 were a gift from the Ludwig Institute for Cancer analyses. Appropriate statistical tests were used to compare data Research (New York, NY), and LS174T dnTCF4 and DLD-1 with similar variances and are referenced in figure legends. dnTCF4 were a gift from Marc van de Wetering (33). Other cell Biological replicates are given as n values. All graphs show lines were from the ATCC. All cell lines were obtained between mean and error bars represent standard deviation (s.d.). Differ- 2009 and 2014, authenticated using short-tandem repeat pro- ences were considered statistically significant when P < 0.05. filing, and tested negative for mycoplasma contamination. P values are given within figures or figure legends. There were Cells were maintained in DMEM supplemented with 10% no missing data. Statistics were calculated with GraphPad Prism FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Bio- or SPSS (IBM). chrom). For doxycycline (DOX, Sigma-Aldrich) induced expression, cells were stimulated with final concentrations of 100 ng/ mL. Polyclonal cell pools for conditional expression were Results generated by transfection of pRTR-SNAIL-VSV or pRTR-ZEB1- PBX3 is overexpressed in colon cancer cells with high WNT VSV using FuGENE 6 (Promega) and selected in 2 mg/mL activity Puromycin (Sigma) for 14 days. WNT stimulation was achiev- To find transcription factors linked to WNT signaling activity in ed using WNT3a (R&D Systems) at a final concentration of colon cancer, we screened previously published gene-expression 20 ng/mL. Transient knockdown was done using predesigned datasets that were derived from human colon cancer cell siRNAs targeting b-catenin (Qiagen), PBX3 (Ambion) or ZEB1 subpopulations with low and high WNT activity (3, 22). Of (Ambion and Dharmacon), or, as control, scrambled siRNA 950 represented genes that encoded for known or putative tran- (siCtrl, Qiagen). hsa-miR-200c (Ambion) was used for transient scription factors, 67 (7.1 %) were significantly (P < 0.05 by t test) miRNA expression. siRNAs and miRNAs were transfected at differentially expressed by 1.25-fold or more. Among those with 10 nmol/L and 30 nmol/L final concentrations, respectively, most significant overexpression in tumor cells with high WNT using HiPerFect (Qiagen). For luciferase reporter assays, cells activity that expectedly included known WNT pathway compo- were transfected or co-transfected in 24-well plates with 10 ng nents or target genes such as LEF1, TCF7, and PROX1,we Renilla luciferase control vector and 100 ng TOPflash or FOP- identified PBX3 (Fig. 1A; Supplementary Table S3). Indeed, in flash luciferase reporter constructs carrying either wild-type or this dataset increased PBX3 expression coincided with high mutant TCF-binding sites (34) in the presence of 0.5 mL expression of WNT pathway components and target genes and,

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A B 5 WNT high WNT low Gene PBX3 D1D2 D1 D2 4 PROX1 PBX3 ) LEF1

( 3 LEF1 DKK1 10 PROM1 2 LGR5

-Log TCF7 EPHB2 CDX2 1 KRT20 MUC2 0 -10123 HiLo Log2 (fold change)

C D PBX3 β-catenin

100

40 β-catenin P < 0.0001 r 20 = 0.52

% Staining intensity

0 0 20 40 60 80 100 PBX3 % Staining intensity

Figure 1. Expression of PBX3 in colon cancer cells with high WNT activity. A, Volcano plot for differentially expressed genes encoding for transcription factors in colon cancer cells with high and low WNT activity (GSE32408 and GSE17375). Colored dots denote genes that are significantly (P < 0.05) upregulated (red) or downregulated (blue) by 1.25-fold or more. Genes are listed in Supplementary Table S3. B, Heat maps of PBX3, selected WNT targets, and differentiation factors in these data sets (D1¼GSE32408, D2¼GSE17375). C and D, Representative (C) immunohistochemistry and (D)quantification of PBX3 and nuclear b-catenin on serial sections of colon cancer. Arrowheads indicate co-staining at the leading tumor edge; scale bar,100mm. For quantification n ¼ 300 cells from six colorectal cancers were scored. P and r values are results of linear-regression analysis.

conversely, with repression of genes associated with a differenti- PBX3 expression is regulated by WNT signaling in colorectal ated tumor cell phenotype (Fig. 1B). We then examined tissue cancer sections of colon cancers and found that PBX3 was heteroge- Next, we determined whether PBX3 expression depended neously expressed in the cytoplasm of tumor cells, with strongest on high WNT activity in colon cancer. Reducing WNT activity by expression at the leading tumor edge, where it overlapped with depletion of b-catenin with two different siRNAs reduced PBX3 on strong expression of nuclear b-catenin, indicating high WNT the protein level in five different colon cancer cell lines with activity (Fig. 1C and D). Interestingly, despite being known as known APC or b-catenin mutations (Fig. 2A; Supplementary a transcription factor, we observed no nuclear staining for PBX3; Fig. S2). We confirmed these effects on the mRNA level by also, not in normal colonic mucosa that showed weak cyto- qRT-PCR in two cell lines, SW1222 and DLD-1, in which b-catenin plasmic positivity only (Supplementary Fig. S1A). To confirm knockdown significantly downregulated PBX3 expression and specificity of PBX3 detection, we therefore examined its expres- that of the WNT target genes AXIN2, NKD1 and LGR5 sion in normal colonic mucosa, primary colon cancer tissue, and (Fig. 2B). Furthermore, we tested the effects of a DOX-inducible colon cancer cell lines by immunoblotting and found that on the dominant negative TCF4 (dnTCF4), a potent inhibitor of the protein level PBX3 isoform A was mainly detected in tumor tissues b-catenin/TCF4 transcription factor complex (33). In LS174T and and also responded to PBX3 knockdown by siRNA (Supplemen- DLD-1 colon cancer cell lines, dnTCF4 induction strongly reduced tary Fig. S1B–S1D). Collectively, these findings indicated upre- transcription from b-catenin/TCF4-binding sites, as seen in TOP- gulation of PBX3 in colorectal cancer cells with high WNT activity flash luciferase reporter assays (Fig. 2C) but also decreased PBX3 on mRNA and protein levels, and suggested a possible regulation protein expression and downregulated PBX3 mRNA levels among of PBX3 by WNT. the panel of WNT target genes (Fig. 2D and E). On the contrary,

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A SW1222 DLD-1 B SW1222 DLD-1 siCtrl + - - + - - siβ-catenin 1 - + - - + - 1.5 siCtrl siβ-catenin 1/2 1.5 siCtrl siβ-catenin 1/2 siβ-catenin 2 -- + -- + *** ****** *** ** *** ******** ** 1 β-Catenin 100 kDa 1 1 0.4 0.3 1 0.6 0.3 0.5 0.5 PBX3 55 kDa 1 0.5 0.3 1 0.5 0.4 0 0 55 kDa Rel. mRNA expression α-Tubulin

AXIN2 NKD1 LGR5 PBX3 AXIN2 NKD1 LGR5 PBX3 CTNNB1 CTNNB1

C LS174T dnTCF4 D LS174T dnTCF4 E LS174T dnTCF4

1.5 - DOX + DOX 1.5 *** - DOX - DOX + DOX ***** *** *** *** + DOX 1 1 PBX3 55 kDa 0.41 0.5 0.5 55 kDa α-Tubulin 0 0

Rel. mRNA expression Rel. luciferase activity TOPflash 1

AXIN2 NKD1 LGR5 PBX3 CTNNB

DLD-1 dnTCF4 DLD-1 dnTCF4 DLD-1 dnTCF4

1.5 - DOX + DOX 1.5 ** ******** ** - DOX - DOX + DOX + DOX 1 1 PBX3 55 kDa 0.61 0.5 55 kDa 0.5 α-Tubulin 0 0

Rel. mRNA expression

Rel. luciferase activity TOPflash 1

AXIN2 NKD1 LGR5 PBX3 CTNNB F G HEK293T HEK293T 4 Ctrl WNT3a Ctrl WNT3a ** *** Active β-catenin 100 kDa 3 * 1 2.7 ** 2 n.s. PBX3 55 kDa 1 6.6 1 55 kDa α-Tubulin Rel. mRNA expression 0 1

AXIN2 NKD1 LGR5 PBX3 CTNNB

Figure 2. Effects of WNT signaling on PBX3 expression in colon cancer cells. A and B, Effects of b-catenin or control (siCtrl) knockdown by siRNA in SW1222 and DLD-1 colon cancer cells, harvested 72 hours after transfection. A, Immunoblotting for indicated proteins on whole-cell lysates. B, qRT-PCR expression analyses for indicated genes. C–E, Induction of a conditional dominant negative TCF4 allele (dnTCF4) by DOX in LS174T and DLD-1 cells. Cells were harvested 24 hours after DOX treatment. C, TOPﬂash dual luciferase assays. D, Immunoblotting for indicated proteins. E, qRT-PCR expression analyses for indicated genes. F and G, Stimulation of HEK293T cells with WNT3a or without stimulation (Ctrl) for 24 hours. F, Immunoblotting for indicated proteins. G, qRT-PCR expression analyses for indicated genes. Numbers below immunoblots indicate normalized fold change by densitometry. Data are mean and error bars indicate s.d. , P < 0.05; , P < 0.01; , P < 0.001; n.s. P > 0.05 by t test; n 3.

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WNT3a stimulation of HEK293T, a human embryonic kidney (Supplementary Fig. S3C). These data suggested that PBX3 is no derived cell line with low intrinsic WNT activity, led to strong direct b-catenin/TCF4 target gene but instead modulated by overexpression of PBX3 and active b-catenin on the protein level, other WNT-dependent downstream factors. as well as upregulation of PBX3 and WNT target gene mRNA (Fig. 2F and G). Taken together, these findings suggested that PBX3 is strongly associated with EMT in colon cancer PBX3 expression is regulated by WNT signaling in colon cancer, Because PBX3 expression was strongest in tumor cells with high whereas this effect is not cell type specific. WNT activity at the infiltrative tumor edge (Fig. 1C), and WNT To test for direct transcriptional regulation by WNT, we then signaling is known to induce EMT in colon cancer (7), we screened the PBX3 promoter sequence and identified three hypothesized that PBX3 might be linked to EMT. To test for a putative b-catenin/TCF4-binding motifs (WWCAAAG; ref. 36) general association of PBX3 with an EMT phenotype, we assem- within 2.5 kb, upstream of the first exon of the PBX3 gene bled and normalized publicly available mRNA expression data of (Supplementary Fig. S3A). To determine whether PBX3 is 856 colon cancers. Gene Set Enrichment Analyses (GSEA) induced by WNT activation via these b-catenin/TCF4 motifs, revealed highly significant (P < 0.001) correlations of PBX3 we subjected 2.5 kb of the PBX3 promoter region, including expression and the expression of two published core EMT gene these motifs or mutated motifs as control, to dual luciferase signatures (Fig. 3A; refs. 29, 30). Moreover, markers that report- reporter assays. Unexpectedly, WNT3a stimulation did not edly indicate EMT in colon cancer were significantly overexpressed increase luciferase expression from the wild-type reporter (Sup- in tumors with high PBX3 levels, among them most prominently plementary Fig. S3B), whereas TOPflash assays confirmed ZEB1 (r ¼ 0.68, P < 0.0001; Fig. 3B). In contrast, CDH1, which strong transcriptional activation of WNT signaling by WNT3a encodes for E-cadherin, negatively correlated with PBX3 in this

A B Mesenchymal transition signatures

Anastassiou VIM

ZEB2

ZEB1

TWIST

SNAI2

SNAIL

CDH1

PBX3 0.8 FN1 Taube 0.6 SNAIL 0.4 FN1 0.2 ZEB2 0.0 VIM

Enrichment Score Enrichment SNAI2 TWIST 0.5 PBX3 positive correlation ZEB1 0.0 PBX3 CDH1 -0.5 PBX3 negative correlation Pearson r Pearson 02,500 7,500 12,500 17,500 -1 +1 Ranked genes

C D PBX3 LAMC2

100 P < 0.0001 r = 0.59 80

LAMC2 40

% Staining intensity

0 200 40 60 80 100 PBX3 % Staining intensity

Figure 3. Association of PBX3 and EMT in colon cancer. A, GSEA for genes ranked by Pearson correlation (Pearson r)toPBX3 expression for two core EMT gene signatures by Anastassiou et al. and Taube et al. (29, 30) in gene-expression datasets of n ¼ 856 colon cancers; P < 0.001. B, Heat map indicates clustering and positive correlation of PBX3 expression with colon cancer relevant EMT markers and negative correlation with CDH1. Colors indicate Pearson r from 1(blue)to1(red). C and D, Representative (C) immunohistochemistry and (D) quantiﬁcation of co-expression for PBX3 and LAMC2 on serial sections of colon cancer. Arrowheads indicate co-staining at the leading tumor edge; scale bar, 100 mm. For quantiﬁcation n ¼ 300 cells from six colorectal cancers were scored. P and r values are results of linear-regression analysis.

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dataset, further supporting the association of PBX3 and EMT. In siRNA. SNAIL expression caused downregulation of E-cadherin addition, to shed more light on the intratumoral distribution of and upregulation of VIM, indicating loss of epithelial and PBX3 and EMT, we assessed colon cancer tissues for PBX3 and gain of mesenchymal features. Depletion of PBX3 partially LAMC2, a factor regulated by ZEB1 (7), by immunohistochem- reversed this effect for E-cadherin but interestingly had no istry. The expression of both markers overlapped at the leading significant effects on VIM expression (Fig. 5G; Supplementary tumor edge (Fig. 3C) and strongly correlated when scored in Fig. S6). Moreover, depleting PBX3 in SW480 and LoVo cell individual colon cancer cells (Fig. 3D). Taken together, these data linescausedupregulationofE-cadherinbutagaindidnot demonstrated that PBX3 is associated with EMT in colon cancer. reduce vimentin expression (Fig. 5H). These data implied that PBX3 expression is at least in part required for the induction PBX3 is required for EMT in colon cancer cells and maintenance of EMT in colon cancer cells. EMT can be induced in colon cancer cells by ectopic expression of SNAIL or ZEB1 (37, 38), and we applied this approach to test PBX3 expression is associated with colon cancer progression whether PBX3 expression is EMT dependent. We used a Because of its dependence on WNT signaling and EMT, both DOX-inducible episomal vector system to overexpress either drivers of colon cancer progression (5), we examined clinical SNAIL or ZEB1 in DLD-1 and LS174T cells, two colon cancer cell associations of PBX3 expression in a collection of 244 colo- lines with low EMT marker expression and pronounced epithelial rectal cancer cases, all of which were UICC stage II with clinical phenotypes (39). In both cell lines, induction of SNAIL caused follow-up records (Supplementary Table S4). We semiquanti- upregulation of PBX3 protein levels within 12 hours (Fig. 4A) and tatively assessed the percentage of PBX3-positive tumor cells, also increased PBX3 mRNA levels together with VIM and ZEB1, and defined scores with complete absence of staining (score 0), while repressing CDH1 and miR-200c (Fig. 4B), indicating an EMT staining in less than 10% of tumor cells (score 1), 10% to 15% phenotype. Immunofluorescence further confirmed upregulation of tumor cells (score 2), and 50% or more tumor cells (score of PBX3 together with increased presence of F-actin stress fibers, 3; Fig. 6A). Of note, although PBX3 staining was strongest in another previously reported feature of EMT (Fig. 4C; ref. 39). tumor cells at the leading tumor edge, it also extended to gland Similarly, ZEB1 induction also caused upregulation of PBX3 forming colon cancer cells in the tumor center, especially in protein and mRNA levels with downregulation of miR-200c in caseswithhighlevelsofPBX3expression(Fig.6A).Kaplan– both cell lines, and CDH1 repression in DLD-1 cells, whereas it Meier statistics revealed similar outcome for cancer-specificand had less effect on the other EMT markers (Supplementary Fig. S4). disease-free patient survival for cases with PBX3 scores 1–3, Because this suggested a regulation of PBX3 through a when compared with cases with PBX3 score 0 (Fig. 6B and C). SNAIL–ZEB1 signaling axis, we next examined the effects of ZEB1 We therefore re-classified into two categories only and found depletion on PBX3 in SW480 and LoVo colon cancer cells, both of that PBX3-positive cases (scores 1–3) showed significantly which have high endogenous levels of ZEB1. ZEB1 depletion by worse cancer-specific and disease-free survival than PBX3 siRNA decreased PBX3 protein and mRNA whereas miR-200c negative (score 0) cases (Fig. 6A–C). We then evaluated co- significantly increased in both cell lines (Fig. 4D and E). We occurrences with other clinical/pathological variables, and confirmed these findings using a second siRNA-depleting observed that PBX3 positivity was more frequent in colorectal ZEB1 in LoVo cells (Supplementary Fig. S5). PBX3 expression cancers of younger patients, whereas there was no significant therefore not only correlates with a mesenchymal phenotype correlation with sex, T-stage, tumor grade, or neo-/adjuvant but also depends on SNAIL/ZEB1-mediated induction of EMT therapy (Supplementary Table S4). Including these variables in colon cancer. into proportional hazards regression analyses demonstrated Because EMT induction by ZEB1 causes repression of independent prognostic power of PBX3 positivity for both miR-200,andPBX3 is a recently identified miR-200 target, we cancer-specific and disease-free survival (Supplementary Tables asked whether the effects of ZEB1 on PBX3 may be indirectly S5 and S6). Because tumor outcome of colon cancer mainly mediated through this miRNA. Using TargetScanHuman (40) depends on distant metastasis, we further investigated PBX3 we identified a highly cross-species conserved 7-mer seed- expression in a second, independent matched case–control matching sequence of miR-200b/c within the PBX3 30-UTR collection of 45 pairs of colon cancers with and without (Fig.5A).TransfectionofSW480 colon cancer cells with synchronous liver metastasis. In this collection, PBX3 positivity miR-200c repressed both ZEB1 and PBX3, with stronger effects was significantly associated with liver metastasis (odds-ratio ¼ on protein than on mRNA levels, as expected for direct miRNA 3.0), further strengthening the link of PBX3 and poor prognosis effects (Fig. 5B and C). We then cloned the 30-UTR of PBX3, (Fig. 6D; Supplementary Table S7). including the miR-200 seed-matching sequence, downstream of Finally, for independent confirmation of these results, we a luciferase open reading frame and found significant down- tested for clinical correlations of PBX3 mRNA levels in the regulation of the reporter activity upon transfection with assembled gene-expression dataset of 856 colon cancer cases, miR-200c (Fig. 5D and E), or upon siRNA mediated knockdown 786 of which had follow-up data on tumor progression. of ZEB1 (Fig. 5F). Both effects were abolished when using a Using ROC curve analyses and Youden's index, we identified luciferase reporter containing the 30-UTR of PBX3 with a an ideal cutoff value at a normalized expression intensity of mutated seed-matching sequence (Fig. 5D–F). These findings 155 (natural scale) of PBX3 mRNA (Supplementary Fig. S7A). demonstrated that PBX3 is targeted by miR-200c and suggested Dichotomal classification of cases by this score revealed a that ZEB1-mediated induction of PBX3 occurs indirectly highly significant positive correlation of high PBX3 expression through de-repression by miR-200c. and poor disease-free survival that also was independent To further determine whether EMT in colon cancer also of other core clinical variables (Fig. 6E; Supplementary depended on PBX3 expression, we induced EMT by SNAIL in Table S8). Of note, the prognostic power of PBX3 was compa- DLD-1 and LS174T cells and concomitantly depleted PBX3 by rable with ZEB1 and outperformed SNAIL in this dataset

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A LS174T/pRTR-SNAIL-VSV B LS174T/pRTR-SNAIL-VSV 0 12 24 48 72 96 (h) DOX 800 *** - DOX SNAIL 760 25 kDa 30 *** + DOX 1 42 50 53 56 61 26 ** PBX3 55 kDa expression 6 1 1.4 1.7 2.0 1.9 1.8 4 *** E-cadherin 130 kDa 2 * ** 1 0.9 0.8 0.7 0.6 0.7

Rel. mRNA 0 α-Tubulin 55 kDa IL 3 H1 A D VIM BX C SN ZEB1 P miR-200c DLD-1/pRTR-SNAIL-VSV DLD-1/pRTR-SNAIL-VSV 0 12 24 48 72 96 (h) DOX 180 *** SNAIL - DOX 25 kDa 160 *** + DOX 1 79 102 112 118 103 4 55 kDa PBX3 expression 3 ** 1 2.8 2.2 2.8 3.0 1.9 2 *** E-cadherin 130 kDa * ** 1 1 0.9 0.7 0.8 0.7 0.4 55 kDa 0 α-Tubulin Rel. mRNA 1 AIL VIM N CDH S ZEB1 PBX3 C miR-200c LS174T/pRTR-SNAIL-VSV DLD-1/pRTR-SNAIL-VSV DAPI GFP F-actin DAPI GFP F-actin

- DOX

+ DOX

DAPI GFP PBX3 DAPI GFP PBX3

- DOX

+ DOX

D E SW480 LoVo SW480 LoVo - - siCtrl + + 6 siCtrl ** 3 siCtrl *** siZEB1 - + - + 4 siZEB1 2 siZEB1 * ** * * ZEB1 1 170 kDa expression 1 0.2 1 0.2 1 PBX3 55 kDa 0.5 1 0.5 1 0.2 α-Tubulin 55 kDa 0 0

Rel. mRNA expression Rel. mRNA 3 B1 X3 X B ZE -200c P ZEB1 PB miR miR-200c

Figure 4. Effects of EMT induction on PBX3 expression in colon cancer cells. A–C, Induction of a conditional SNAIL allele in LS174T and DLD-1 cells by DOX. A, Immunoblotting for indicated proteins at indicated time points after DOX treatment. B, Gene-expression analyses by qRT-PCR for indicated genes after 72 hours with or without DOX treatment. C, Representative confocal immunoﬂuorescence for indicated proteins after 72 hours with or without DOX treatment; scale bars, 50 mm. D and E, Effects of ZEB1 or control (siCtrl) knockdown by siRNA in SW480 and LoVo colon cancer cells, harvested 72 hours after transfection. D, Immunoblotting of indicated proteins on whole-cell lysates. E, Gene-expression analyses by qRT-PCR for indicated genes. Numbers below immunoblots indicate normalized fold change by densitometry. Data are mean and error bars indicate s.d. , P < 0.05; , P < 0.01; , P < 0.001 by t test; n 3.

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PBX3 Links EMT and Poor Prognosis in Colon Cancer

A B

miR-200c SW480 Human miR-200c Ctrl Chimpanzee Mouse ZEB1 170 kDa Rabbit 1 0.19 Dog 55 kDa Horse PBX3 Cow 1 0.48 α-Tubulin 55 kDa

PBX3

3' UTR C SW480 D 1.5 Ctrl miR-200c * * PBX3 WT 1 miR-200c

expression

0.5 PBX3 MUT

Rel. mRNA ZEB1 PBX3 E F SW480 SW480 1.5 Ctrl siCtrl n.s. ** n.s. 1.5 miR-200c n.s. *** n.s. siZEB1 1 1

0.5 0.5

Rel. luciferase activity 0 Rel. luciferase activity 0 pGL3 PBX3 WT PBX3 MUT pGL3 PBX3 WT PBX3 MUT

G H LS174T/pRTR-SNAIL-VSV DLD-1/pRTR-SNAIL-VSV SW480 LoVo

siCtrl + + - ++- siCtrl + - + - siPBX3 - - + --+ siPBX3 - ++- - DOX - + + ++ PBX3 55 kDa SNAIL 25 kDa 1 0.3 10.7 SNAIL 25 kDa PBX3 55 kDa 1 0.6 10.7 12.40.5 1 2.8 1.5 E-cadherin 130 kDa E-cadherin 130 kDa 1 1.7 11.8 10.10.3 1 0.3 0.6 Vimentin 55 kDa α-Tubulin 55 kDa 1 1.2 11.0 α-Tubulin 55 kDa

Figure 5. Modulation of PBX3 expression by miR-200c and requirement of PBX3 for EMT in colon cancer cells. A, Illustration of the PBX3 30-UTR indicating phylogenetic conservation of a miR-200 seed-matching sequence. B and C, Transfection of SW480 cells with miR-200c or non-targeting (Ctrl) oligonucleotides for 48 hours. B, Immunoblotting for indicated proteins on whole cell lysates. C, qRT-PCR expression analyses for indicated genes. D, 30-UTRs of PBX3 with wild-type (WT) and mutated (MUT) miR-200 seed matching sequences. E and F, Effects on dual luciferase reporter assays in SW480 cells transfected with pGL3 carrying WT or MUT 30-UTRs of PBX3, or with empty vector (pGL3). E, Co-transfection of miR-200c or non-targeting (Ctrl) oligonucleotides for 72 hours. F, Co-transfection of ZEB1 or control (siCtrl) siRNA for 72 hours. G, Induction of a conditional SNAIL allele in LS174T and DLD-1 cells by DOX and immunoblotting for indicated proteins on whole-cell lysates after PBX3 or control (siCtrl) knockdown by siRNA for 72 hours. H, Immunoblotting for indicated proteins on whole cell lysates of SW480 and LoVo colon cancer cells after PBX3 or control (siCtrl) knockdown by siRNA for 72 hours. Numbers below immunoblots indicate normalized fold change by densitometry. Data are mean and error bars indicate s.d.; , P < 0.05; , P < 0.01; , P < 0.001; n.s. P > 0.05 by t test; n 3.

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A PBX3 negative PBX3 positive Score 0 (n = 54) Score 1 (n = 68) Score 2 (n = 88) Score 3 (n = 34)

B C

1.0 1.0 6/54 1.0 1.0 9/54 0.8 0.8 0.8 0.8 0.6 0.6 0.6 0.6 46/190 0.4 PBX3 score 0.4 0.4 PBX3 score 0.4 67/190 0 PBX3 0 PBX3 0.2 1 0.2 0.2 1 0.2 2 negative HR=2.6 2 negative HR=2.7 P P P 0 3 = 0.11 0 positive = 0.023 Disease-free survival 0 3 P = 0.031 0 positive = 0.004

Cancer-specific survival 02.557.510 12.5 02.557.510 12.5 02.557.510 12.5 02.557.510 12.5

Years after surgery Years after surgery

D E GSE14333 GSE39582 Combined P = 0.011 80 1.0 1.0 1.0 13/95 57/283 60 0.8 0.8 44/188 0.8 0.6 0.6 0.6 40 133/369 173/503 0.4 40/134 0.4 0.4 20 PBX3 0.2 0.2 0.2 % Metastasized low HR=2.2 HR=1.6 HR=1.8 P P P 0 Disease-free survival 0 high = 0.013 0 = 0.004 0 < 0.0001 PBX3 PBX3 0 5 10 15 0 5 1510 0 5 10 15 Negative Positive Years after surgery

Figure 6. PBX3 expression in colorectal cancer indicates poor prognosis. A, Assessment of PBX3 immunostaining in primary human colorectal cancers. Tumors were assigned semiquantitative expression scores from 0 (no staining) to 3 (strong staining) and accordingly categorized as PBX3 negative (score 0) and positive (scores 1–3). Arrows indicate stained tumor cells. Case numbers per score are indicated; scale bars, 100 mm. B and C, Kaplan–Meier plots for different PBX3 expression scores and categories in a collection of n ¼ 244 stage II colorectal cancers for (B)cancer-speciﬁc survival and (C)disease-free survival. HR, hazard ratio for PBX3-positive cases. P values are log-rank test results. Ratios on curves indicate the number of events over the number of patients per group. D, Association of PBX3 expression and liver metastasis in a matched case control collection of colon cancers. The P value is c2 test result. E, PBX3 mRNA expression and survival association in two individual and combined datasets of a total of n ¼ 786 colon cancers. GEO accession numbers of individual datasets are indicated. Kaplan–Meier plots for cases with low and high PBX3 expression. HR for PBX3 high cases. P values are log-rank test results. Ratios on curves indicate the number of events over the number of patients per group.

(Supplementary Fig. S7B; Supplementary Tables S9 and S10). Discussion Collectively, these ﬁndings suggest that PBX3 is associated with tumor progression and poor survival in patients with The ability of epithelial cancer cells to loose cellular junc- ﬁ colorectal cancer. tions and polarity with subsequent in ltration of tumor

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PBX3 Links EMT and Poor Prognosis in Colon Cancer

surrounding stromal tissue is a main aspect of EMT and with colorectal cancer. PBX3 expression was significantly associ- hallmark of cancer invasion (41). Here, we identify strong ated with poor cancer-specific survival and strongly correlated overexpression of PBX3 in tumor cells with high WNT activity with an increased risk for cancer progression in a collection of undergoing EMT at the leading tumor edge of colorectal cancer. 244 stage II colorectal cancers, whereas this was independent We demonstrate that PBX3 expression is induced in this tumor of other core clinical variables. Stage II colorectal cancer is char- cell subset by WNT and the EMT regulating transcription factors acterized by local disease with full-thickness involvement of SNAIL and ZEB1, whereas this induction—at least partially— the bowel wall but absence of lymphatic or distant metastasis occurs indirectly through a decreased repression of PBX3 (48). Accordingly, most of these patients can be cured by surgical mRNA by miR-200.Thesefindings are in agreement with recent resection alone. However, disease progression after surgery still data that demonstrated targetability of PBX3 by different is observed in 25% to 30% of these cases, and patients may microRNAs (16), and therefore place its expression in colon eventually die from their disease (49). We therefore suggest that cancer within a WNT and EMT regulatory network (42). Fur- assessing PBX3 expression may be promising to identify poten- thermore, we demonstrate that PBX3 expression is required for tially aggressive cases of stage II colorectal cancer that may benefit a full EMT phenotype in colon cancer cells, since its depletion from adjuvant therapy and increased clinical attention despite partially blocked EMT induction by ZEB1 and SNAIL, and low tumor stage (50). Moreover, PBX3 expression also signifi- increased the expression of E-cadherin, indicating a shift cantly correlated with metastasis in our case–control collection, toward more epithelial differentiation. However, because PBX3 and thus also may indicate progression in late-stage disease. In depletion had no significant effects on vimentin expression, we addition, we found that on the mRNA level PBX3 expression suggest that only certain aspects of EMT depend on PBX3. was highly significantly associated with poor outcome in a com- Nevertheless, considering recent findings that PBX3 induces bined dataset with clinical information on 786 colon cancers, EMT in gastric cancer cells (15, 43), this indicates that it may including all stages. This not only supported the findings in generally be involved in EMT regulation in gastrointestinal our tissue collections but also confirmed results from a previous cancers. Because PBX3 also has been shown to increase migra- study that suggested an association of PBX3 mRNA expression and tion and invasion of colon cancer cells (14) both of which are poor patient survival in a smaller patient series (14). However, features of EMT, this further supports the notion that PBX3 before PBX3 expression analysis may be introduced into routine directly contributes to the infiltrative phenotype of colon pathology workup of colorectal cancer specimens, robust multi- cancer cells at the leading tumor edge. However, the exact center validation studies in prospectively recruited patient series mechanism by which PBX3 influences EMT in colon cancer will be required to determine its true prognostic biomarker still remains to be determined, keeping in mind that different potential. If the independent prognostic power of PBX3 can be isoforms of PBX3 and in some organs variable intracellular validated, we suggest that this may be due to gauging EMT, which distributions are known and that PBX3 may function as cofac- currently is not sufficiently reflected by other routinely assessed tor for other homeobox proteins (11, 44). clinical and pathological variables. In primary colon cancer tissues, we found that PBX3 can easily be visualized in situ by immunostaining. Importantly, Disclosure of Potential Conflicts of Interest labelling in these tumors was restricted to cancer cells while No potential conflicts of interest were disclosed. tumor surrounding stromal cells were PBX3 negative. Given that markers which robustly indicate EMT in colon cancer are Authors' Contributions scarce, and detection of ZEB1, SNAIL and vimentin can be Conception and design: S. Lamprecht, M. Kaller, D. Horst difficult and confounded by labelling of stromal cells (45–47), Development of methodology: S. Lamprecht, M. Kaller, H. Hermeking, we propose that PBX3 may be a useful marker to highlight and D. Horst Acquisition of data, analysis and interpretation of data, writing, review, further study colon cancer cells undergoing EMT in situ.Fur- PBX3 and/or revision of the manuscript, and administrative, technical, or material thermore, we demonstrate that mRNA levels strongly support: S. Lamprecht, M. Kaller, E.M. Schmidt, C. Blaj, T.S. Schiergens, J. Engel, correlated with EMT in a large gene-expression dataset derived A. Jung, H. Hermeking, T.G.P. Grunewald,€ T. Kirchner, D. Horst from 856 colon cancer samples. Considering the restriction of Study supervision: D. Horst PBX3 expression to cancer cells, we therefore propose that on the gene-expression level PBX3 may indicate the overall degree Acknowledgments of EMT in colon cancer specimens with little confounding by This study was supported by grants from the Deutsche Krebshilfe (11169; theamountofstromaltissuewithineachsample.Ofnote, to D. Horst), the Wilhelm Sander-Stiftung (2012.031; to D. Horst), and the Rudolf Bartling Stiftung (to D. Horst and H. Hermeking). T.G.P. Grunewald€ PBX3 expression was not completely restricted to infiltrative was supported by grants from the Deutsche Krebshilfe (111886 and tumor cells at the leading tumor edge but also extended to 70112257). We are grateful to A. Heier, J. Kovi,€ M. Melz, and A. Sendelhofert glandular differentiated colon cancer cells, especially in cases for experimental assistance. with high levels of PBX3 expression. Because similar observa- tions also were made for ZEB1 and SNAIL (45, 47), it remains The costs of publication of this article were defrayed in part by the to be determined to what extent infiltrative tumor cell mor- payment of page charges. This article must therefore be hereby marked phology and such gradually expressed EMT-related factors advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate indicate identical or only partially overlapping colon cancer this fact. cell subpopulations. Our findings in patient series with clinical follow-up data Received September 3, 2017; revised January 2, 2018; accepted January 25, suggest that PBX3 expression is linked to poor outcome in patients 2018; published first February 1, 2018.

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PBX3 Is Part of an EMT Regulatory Network and Indicates Poor Outcome in Colorectal Cancer

Sebastian Lamprecht, Markus Kaller, Eva Marina Schmidt, et al.

Clin Cancer Res Published OnlineFirst February 1, 2018.

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