Expression of the PTTG1 Oncogene Is Associated with Aggressive Clear Cell Renal Cell Carcinoma

Published OnlineFirst July 17, 2012; DOI: 10.1158/0008-5472.CAN-11-2330

Cancer Molecular and Cellular Pathobiology Research

Bill Wondergem1, Zhongfa Zhang1, Dachuan Huang5, Choon Kiat Ong5, Julie Koeman2, David Van't Hof3, David Petillo1, Aikseng Ooi1, John Anema4, Brian Lane4, Richard J. Kahnoski4, Kyle A. Furge1,3, and Bin Tean Teh1,5

Abstract The pituitary tumor transforming gene (PTTG1) is a recently discovered oncogene implicated in malignant progression of both endocrine and nonendocrine malignancies. Clear cell renal cell carcinoma (ccRCC) is cytogenetically characterized by chromosome 3p deletions that harbor the ccRCC-related von Hippel-Lindau, PBRM1, BAP1, and SETD2 tumor suppressor genes, along with chromosome 5q amplifications where the significance has been unclear. PTTG1 localizes to the chromosome 5q region where amplifications occur in ccRCC. In this study, we report a functional role for PTTG1 in ccRCC tumorigenesis. PTTG1 was amplified in ccRCC, overexpressed in tumor tissue, and associated with high-grade tumor cells and poor patient prognosis. In preclinical models, PTTG1 ablation reduced tumorigenesis and invasion. An analysis of gene expression affected by PTTG1 indicated an association with invasive and metastatic disease. PTTG1-dependent expression of the RhoGEF proto-oncogene ECT2 was observed in a number of ccRCC cell lines. Moreover, ECT2 expression correlated with PTTG1 expression and poor clinical features. Together, our findings reveal features of PTTG1 that are consistent with its identification of an oncogene amplified on chromsome 5q in ccRCC, where it may offer a novel therapeutic target of pathologic significance in this disease. Cancer Res; 72(17); 4361–71. 2012 AACR.

Introduction frequently reported genetic abnormality in ccRCC, occurring in Renal cell carcinoma (RCC) is the most common type of the majority of cases (4, 5) and leading to stabilization of the kidney cancer and accounts for approximately 39,000 new HIF proteins and subsequent transcription of HIF target genes – fi cases and 13,000 deaths per year within the United States (6 8). Ampli cation of chromosome 5q is the second most (1). Clear cell renal cell carcinoma (ccRCC) is the most com- common cytogenetic change associated with ccRCC, yet the fi – mon subtype of renal cell carcinoma and accounts for approx- signi cance of this abnormality is not understood (3, 9 11). imately 75–80% of cases (2). ccRCC is characterized by a Multiple cytogenetic studies have concluded that genetic – recurrent set of chromosomal abnormalities that includes alterations of chromosome 5q, particularly 5q33.2 35, are – deletions of chromosomes 3p, 9q, and 14q and amplifications associated with the progression of ccRCC (10, 12 15). Despite of chromosome 5q and 7 (3). Deletion of chromosome 3p the frequency of this aberration, few candidate oncogenes removes one allele of the von Hippel-Lindau (VHL) tumor within this region of copy number gain have been implicated suppressor gene, whereas somatic mutation typically inacti- in ccRCC (16). vates the remaining allele. Biallelic loss of VHL is the most Pituitary tumor transforming gene (PTTG1) is a recently discovered oncogene that resides on 5q33.3 and has been implicated in the development and progression of many 1 2 Authors' Affiliations: Laboratories of Cancer Genetics, Cytogenetics, malignancies (17). Upregulation of PTTG1 has been correlated and 3Computational Biology, Van Andel Research Institute; 4Department of Urology, Spectrum Health Hospital, Grand Rapids, Michigan; and 5NCCS- with aggressive disease and poor prognosis in hepatocellular VARI Translational Research Laboratory, National Cancer Centre Singa- carcinoma, prostate cancer, esophageal cancer, and glioma, pore, Singapore among others (18–22). PTTG1 overexpression has also been Note: Supplementary data for this article are available at Cancer Research associated with the invasiveness of thyroid and colorectal Online (http://cancerres.aacrjournals.org/). cancers (23, 24). The oncogenic potential of PTTG1 was initially Current address for Z. Zhang: Center for Systems and Computational shown by its ability to transform NIH3T3 fibroblasts upon Biology, The Wistar Institute, Philadelphia, Pennsylvania; and current overexpression. This was later substantiated in HEK293 cells in address for J. Koeman, Laboratory of Genome Biology, Van Andel Research Institute, Grand Rapids, Michigan. which overexpression of PTTG1 resulted in increased cell proliferation, anchorage-independent growth in soft agar, and Corresponding Author: Bin Tean Teh, Laboratory of Cancer Gene- tics, Van Andel Research Institute, 333 Bostwick Avenue NE, Grand tumor formation in nude mice (25). Although its potent Rapids, MI 49503. Phone: 616-234-5296; Fax: 616-234-5297; E-mail: transforming ability is now well established both in vitro and [email protected] in vivo, its precise oncogenic mechanism remains unclear as doi: 10.1158/0008-5472.CAN-11-2330 PTTG1 seems to be a multi-functional protein with several 2012 American Association for Cancer Research. possible means of promoting tumorigenesis.

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As a securin protein, PTTG1 regulates sister chromatid out. The gene expression values were turned into dichotomous separation during mitosis and overexpression can interfere variables (high and low) using an optimal cut procedure (see with this process, leading to aneuploidy (26). Other studies Supplementary Methods). To identify differentially expressed have suggested an important role for PTTG1 in influencing the genes following PTTG1 knockdown, expression profiles from tumor microenvironment, in part by increasing the expression treated and control samples were generated from the Affyme- and secretion of the important proangiogenic factors (25, 27– trix HG-U133 Plus 2.0 using RMA analysis and data were 29). PTTG1 involvement in cell-cycle progression and prolif- subsequently unlogged. Genes with expression values less than eration, p53-mediated apoptosis, and DNA repair has also been 20 were considered to be not expressed in the indicated cell reported (27, 30–32). The key to PTTG1 oncogenic effects likely line. Fold-change of each gene was defined to be the ratio of lies in its role as a transcriptional activator. In 2000, Pei and expression intensity of that gene in the treated sample over colleagues showed that the activation of the MAPK pathway that in the control sample, if the ratio was positive; otherwise, led to phosphorylation of the PTTG1 transactivation domain, the fold change was defined to be the negative of inverse value initiating PTTG1 nuclear translocation and transcriptional of the ratio. The gene expression data have been deposited at activity (33), and direct downstream targets identified thus the Gene Expression Omnibus (GEO 25493). far include basic fibroblast growth factor and c-MYC (27, 34). Mutation of the double PXXP motif required for transactiva- Fluorescent in situ hybridization tion has been shown to abrogate the transforming effects of FISH probes were prepared from purified BAC clones CTD- PTTG1 expression (17, 35, 36). 2155B8 (5q33.3; Invitrogen) and RP11-254G13 (5q11.2; BAC- Although overexpression of PTTG1 has now been observed PAC Resource Center, bacpac.chori.org). The BAC clone DNA in many cancer types, there is little information about the role was labeled with SpectrumGreen or SpectrumOrange (Abbott of the PTTG1 oncogene in kidney cancer. Here, we show that Molecular Inc.), by nick translation. Tumor touch preparations PTTG1 overexpression in ccRCC is associated with patient were prepared by imprinting thawed tumors onto glass slides. survival and assess its putative role as a promoter of disease Images were analyzed using FISHView EXPO v6.0 software progression. In addition, we show that this 5q oncogene is (ASI) and 100 to 200 interphase nuclei were scored for each genetically amplified in ccRCC and contributes to the onco- sample. genic transformation of renal cancer cells, as well as, the progression of ccRCC from localized to invasive disease. We Immunohistochemistry also show that PTTG1 regulates the expression of the Rho-GEF Formalin-fixed paraffin-embedded tissues were microtome ECT2, another novel proto-oncogene that exhibits significant cut into 3-micron sections, deparaffinized, and rehydrated. overexpression in aggressive ccRCC. Samples were boiled in citrate buffer, cooled, and subsequently incubated with 3% hydrogen peroxide to quench endogenous Materials and Methods peroxidase activity. The sections were blocked with goat serum DNA copy number analysis and incubated with human anti-PTTG1 antibody (Invitrogen) Tissue was collected from Spectrum Health Hospital of or anti-ECT2 antibody (Sigma Aldrich) overnight followed by Grand Rapids, MI, and the Cooperative Human Tissue incubation with biotinylated anti-rabbit IgG for 1 hour at room Network under the VARI Institutional Review Board. All temperature. Subsequently, the slides were stained with an tissue was snap-frozen in liquid nitrogen immediately after avidin–biotin complex to visualize antigen–antibody complex, nephrectomy and stored at 80C. Genomic DNA was counterstained with hematoxylin, dehydrated, and mounted. extracted using a Jetquick Maxiprep Kit (Genomed) per the Substitution of TBS for primary antibody was used as a manufacturer's instructions. Single nucleotide polymor- negative control. Sections were imaged and sent to pathologist phism (SNP) mapping assay was carried out according to (Dr. James Resau) for semiquantitative analysis. Stain intensity, Affymetrix's protocol. The SNP intensity data were analyzed either nuclear or cytoplasmic, was scored on a scale of 0 (low) using Affymetrix GeneChip Genotyping analysis software to 4 (high). Statistical significance of variable stain intensity (GTYPE) version 4.0 and examined largely with the excep- between low-grade ccRCC (Fuhrman grades 1 and 2) and high- tion that a divide-and-conquer algorithm was used to seg- grade ccRCC (Fuhrman grades 3 and 4) was assessed by ment the copy number data (see Supplementary Methods; Wilcoxon rank-sum test. ref. 37). The SNP intensity data have been deposited at the Gene Expression Omnibus (GEO 25399). Cell culture Caki-1, ACHN, A-498, HK2, SW-156, and 786-O RCC cell lines PTTG1 and ECT2 expression analysis were obtained from the American Type Culture Collection Expression data were generated from the Affymetrix HG- (ATCC). SN12C cells were kindly provided by Dr. George Vande U133 Plus 2.0 platform as previously described (15). A Cox Woude (Van Andel Research Institute). UO-31, TK-10, and Proportional Hazards model using cancer-specific survival and RXF393 cells were obtained from the National Cancer Institute. the expression level of each examined gene as a continuous HK2 cell lines were obtained in November 2011 and charac- predictor. The significance values (P values) from the Cox terized by short-tandem repeat analysis by ATCC. All other cell models are reported in the figures and the text. To visualize lines were obtained before the new requirements for publica- the relationship between gene expression levels and cancer- tion and; therefore, have not been authenticated. Cells were specific survival, Kaplan–Meier survival analysis was carried maintained in Dulbecco's Modified Eagle Medium (DMEM;

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Invitrogen) supplemented with 10% FBS (Invitrogen), in a Plasmid construction humidified incubator containing 5% CO2 at 37 C. Human PTTG1 from a human cDNA pool (Clontech) was amplified umbilical vascular endothelial cells (HUVEC) were obtained by PCR using forward primer (50-CTCGGATCCACCATGGC- from Clonetics and maintained in Clonetics EBM-2 medium TACTCTGATCTATG-30) and reverse primer (50-AGA ATTC- supplemented with EGM-2 SingleQuots or EGM-MV Single- CAAATATCTATGTCACAGCAAACAGG-30). The resulting PT- Quots (Lonza). TG1 PCR product was inserted into pcDNA6/myc-His B vector (Invitrogen) at the Bam HI and Eco RI restriction sites. Immunoblotting Total protein was extracted from cell lines using HNTG Immunofluorescence lysis buffer supplemented with protease-inhibitor cocktail Cells were fixed and permeabilized using a 1:1 methanol/ (Roche). Briefly, cells were scraped and incubated on ice in acetone solution at 20C for 3 minutes and subsequently lysis buffer for 30 minutes and subsequently centrifuged at blocked with 10% donkey serum in PBS with 1% bovine serum 20,000 rpm for 20 minutes at 4C to isolate soluble proteins. albumin for 1 hour. Cells were then incubated with rabbit This fraction was heat denatured and used for SDS-PAGE polyclonal anti-PTTG1 antibody (Invitrogen) or anti-b-actin and subsequent transfer to a polyvinylidene difluoride mem- antibody (Abcam) diluted 1:100 in PBS with 1% bovine serum brane. Target proteins were identified using rabbit polyclon- albumin for 2 hours at room temperature. Cells were then al anti-PTTG1 (Invitrogen), mouse monoclonal anti-b-Actin incubated with FITC-conjugated and TRITC-conjugated sec- (Abcam) or rabbit polyclonal anti-ECT2 (Santa Cruz Bio- ondary antibodies (Cell Signaling), counterstained with DAPI, technology) primary antibodies, and appropriate secondary and imaged. reagents. Colony formation assay Quantitative real-time PCR Cells with or without PTTG1 were tested for their anchor- RNA was isolated from cells using Trizol Reagent (Invitro- age-independent growth potential. Parental lines 786-O and gen) using a standard guanidinium thiocyanate-phenol-chlo- SN12C and cell lines transduced with shPTTG1 or control roform extraction method. Taq-Man probes specific for vector were counted and 1 103 cells were placed in 2 PTTG1, ECT2, and b-actin were obtained from Applied Bio- DMEM, 20% FBS, mixed with equal volume 0.7% agarose, and systems. RNA was reverse transcribed and amplified for quan- plated on top of a base layer of 0.8% agar and DMEM, 10% FBS titative real-time PCR using the qScript One-Step SYBR Green in a single well of a 6-well plate. For each cell line, 6 replicates qRT-PCR Kit (Quanta Biosciences) according to protocol. were plated. Cells were incubated at 37C and fed DMEM with Analysis was carried out using the comparative-Ct method 20% FBS twice weekly for 3 weeks until colonies became visible. normalizing target mRNA levels to b-actin mRNA. A control The plates were methanol fixed and stained in crystal violet for human RNA sample (Applied Biosystems) was amplified and imaging. used as a calibrator for this analysis. Subcutaneous xenograft RNA interference Cells with and without PTTG1 knockdown were tested for PTTG1 was transiently silenced in vitro using two separate their tumorigenic ability in nude mice. Two different parental siRNAs (Invitrogen), and ECT2 was silenced using a combina- lines, 786-O and SN12C, and their derivatives shPTTG1-786-O, tion of 3 siRNAs (Invitrogen). A nontargeting siRNA (Invitro- con shPTTG1-SN12C, or control-vector transduced cells were gen) was used as a negative control. SiRNA was transfected into cultured and 3 106 resuspended in 0.2 mL DMEM with 10% ccRCC cell lines with lipofectamine-2000 (Invitrogen) using FBS were injected subcutaneously into the right flank of each standard lipid transfection methods. Cells were allowed to animal. Tumor volume was caliper measured every other day recover from lipid reagent permeabilization following trans- and animal weight was measured weekly until tumor burden fection by incubation in DMEM 10% FBS overnight before necessitated sacrifice. usage. Stable knockdown of PTTG1 was achieved using the pLKO.1 lentivirus-based short hairpin RNA (shRNA) deliv- Proliferation assays ery system (38, 39). Plasmids carrying shRNA sequence against Cells were seeded into 96-well plates at an approximate PTTG1 (clones TRCN0000015104, TRCN0000015105, TRCN- concentration of 500 cells per well, 12 wells per treatment. The 0000015106, TRCN0000015103, TRCN0000015107) were pur- cells were allowed to adhere overnight before cell viability was chased from Openbiosystem (Thermo Scientific), whereas a determined by the colorimetric 3-(4,5-dimethylthiazol-2yl)-5- negative control pLKO.1 vector carrying a nontargeting (3-carboxymethoxyphenyl)-(4-sulfophenyl)-2H-tetrazoluim shRNA sequence was purchased from Addgene. The genera- assay according to the manufacturer's instruction (MTS; Pro- tion of lentivirus particles was achieved by cotransfecting mega). This was repeated every 24 hours for 4 days. The results pLKO.1clone with second generation packaging vectors, are presented as fold change from initial measurement (t ¼ 0). psPAX2 (Addgene plasmid 12260), and pMD2.G (Addgene plasmid 12259) into HEK293FT cells (Invitrogen). Virus pack- Invasion assays aging, transduction, and selection of stably transduced cells Cells transfected with anti-PTTG1 siRNA or a nontargeting were done according to a manufacturer recommended proto- control siRNA were serum starved for 24 hours in culture. col (Addgene). Subsequently, 5 104 (1 105 for Caki-1 cells) were

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resuspended in 200 mL serum-free-media and placed onto same frequency of amplification observed in our SNP array BioCoat Growth Factor Reduced Matrigel invasion chambers study. To further investigate copy number gain at the PTTG1 (BD Biosciences; Pharmingen), which were then placed into a locus at 5q33.3, interphase FISH was carried out on ccRCC 24-well plate containing 800 mL DMEM plus 10% FBS as a tumor tissue touch-prep samples (Supplementary Table chemoattractant. Cells were allowed to invade through the S2, Fig. 1D). FISH analysis showed between 2 and 9 copies of chambers for 16 hours before the chambers were swabbed and the PTTG1 gene in ccRCC samples, confirming frequent invading cells were methanol fixed and stained with crystal amplification at this locus. More than 2 copies of the PTTG1 violet for imaging and counting. Two replicates per treatment oncogene were observed in 9 of 11 tumors tested by FISH. Some were used and the experiment was carried out in duplicate. tumors showed large-scale amplification, as detected by increased copy number of a control probe located at 5q11.2 ECT2 rescue experiment immediately adjacent to the centromere. In contrast, other HK-2 cells were transfected with empty vector pcDNA6 or tumors showed increased copy number of only the PTTG1 PTTG1 construct. The stable transfected cells were established probe at 5q33.3, indicative of smaller amplification events and mantained in 20 mg/mL blasticidin (Sigma). Then the (Fig. 1D). stable cell lines were transduced with lentivirus expressing shRNA targeting ECT2 or a control construct prior subjected to PTTG1 is overexpressed in ccRCC at both the mRNA and cell proliferation assay and colony formation assay. protein levels Next, we examined the expression of PTTG1 in ccRCC. We Results observed a significant increase in PTTG1 mRNA levels in 5q amplification results in frequent PTTG1 copy number carcinoma tissue specimens relative to nondiseased kidney gain as measured by gene expression microarrays (Fig. 1B and C and The SNP arrays were used to identify regions of copy number Supplementary Fig. S1). Elevated PTTG1 mRNA expression is change associated with the pathogenesis of ccRCC (37). Con- associated with high grade (Fuhrman grade 3 and 4) versus low sistent with previous reports, we observed deletion of chro- grade (Fuhrman grade 1 and 2; P ¼ 0.04), high tumor stage (P ¼ mosome 3p to be the most frequent cytogenetic abnormality in 0.002), and decreased patient postoperative survival (P ¼ ccRCC. Deletion of some portion of chromosome 3p was seen 0.008). Expression of PTTG1 does not follow a bi-model in 39 of 43 (91%) cases tested by SNP 100 K microarray (data not expression pattern in which a subset of samples contains shown), a frequency consistent with previous reports (12, 37). extremely high levels of PTTG1 and is associated with The second most common chromosomal aberration was decreased survival rates. Rather, increased expression of amplification of the long arm of chromosome 5 (Fig. 1A). We PTTG1 is associated with a proportional decrease in survival observed copy number gain of this region in 26 of 43 (60%) probability (Supplementary Fig. S1), suggesting PTTG1 plays a cases and identified 5q33.2–35 as a refined region of copy role in the progression of this disease and the acquisition of an number gain, amplified in all cases showing 5q amplification. aggressive phenotype. Similar results about the minimal region of amplification have PTTG1 overexpression was confirmed by immunohisto- been obtained by others using similar SNP profiling studies chemical staining of ccRCC tumors and adjacent normal (16, 40). Notably, we observed a high degree of variability in kidney (Fig. 2). Elevated PTTG1 protein levels were seen in amplicon size. Some cases showed gain of all of chromosome 5, ccRCC sections relative to normal kidney with the strongest whereas other events were restricted to the terminal end of staining observed in high-grade tumors. There was a signifi- chromosome 5q. Polyploidy of chromosome 5 was observed in cant difference (P < 0.05) in staining intensity between low- 9 of 43 tumors. Smaller amplification events, typically involving grade tumors (Fuhrman grades 1 and 2) and high-grade tumors approximately half of 5q, were more common than whole (Fuhrman grades 3 and 4). These findings are consistent with chromosome gain. The smallest amplicon detected by SNP recent reports of PTTG1 gene and protein expression patterns microarray showed copy number gain from 5q33-5qter, span- that have identified PTTG1 in a gene signature of ccRCC ning just 3 cytobands. In all cases with 5q gain, PTTG1 is pulmonary metastases and showed that PTTG1 protein located within the minimal region of amplification (Fig. 1A). expression is associated with high nuclear grade, tumor stage, We noted that the oncogenes, EGR1 and CSF1R, are located in and tumor size (41, 42). Furthermore, we observed predomi- the region of PTTG1 amplification and are overexpressed in nately nuclear staining with some high-grade cases exhibiting ccRCC. However, expression of these genes is not associated both strong nuclear and cytoplasmic staining, consistent with with overall survival (Supplementary Table S1 and data not a previous report of both PTTG1 nuclear positivity and cyto- shown). plasmic mislocalization in malignant esophageal squamous As PTTG1 is a known oncogene that maps within the region cell carcinoma (ESCC) tissues (22). of chromosome 5q amplification, we examined the copy number status and expression levels of this gene in ccRCC in greater PTTG1 influences ccRCC tumorigenesis both in vitro and detail. Examination of the copy number status of PTTG1 locus in vivo using the Broad Institute's Genomic Identification of Signifi- In order to study the importance of the PTTG1 oncogene cant Targets in Cancer (GISTIC; http://www.broadinstitute. in vitro and in vivo, we examined the level of PTTG1 protein org/igv/GISTIC) module shows copy number gain at the expression in several ccRCC and other kidney-derived cell PTTG1 locus in 60% of cases tested (data not shown), the lines (Fig. 3A). Compared with tumor-derived cell lines,

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Figure 1. The PTTG1 gene locus is amplified in ccRCC. A, a heatmap reflecting chromosome 5 copy number in ccRCC (n ¼ 43). White color indicates equal copy number as compared with normal tissue; the red and blue colors are reflective of copy number gain and loss, respectively, compared with normal tissue. The intensity of the color indicates significance of copy number increase or decrease as described in Materials and Methods. The location of the PTTG1 FISH probe and the control FISH probe used in Fig. 1D is also shown. B,expression levels of PTTG1 as determined by gene expression microarray. Nondiseased samples (N) and ccRCC tumors as shown. The tumor samples are grouped by grade (1–4). Increased PTTG1 expression is associated with high grade (Fuhrman grade 3 and 4) versus low grade (Fuhrman grade 1 and 2; P < 0.05). Significance was determined using a Wilcox rank sum test. C, Kaplan–Meier curve of PTTG1 expression and cancer-specific survival in ccRCC samples partitioned into high and low groups as described in Materials and Methods. D, representative FISH images of samples described in Supplementary Table S1. False color images of DAPI counterstaining (blue), a PTTG1 locus-specific probe (red), and a chromosome 5 centromeric probe (green) are shown.

nontransformed HUVEC and HK2 immortalized renal prox- positive cell lines, SN12C and 786-O, were selected for imal tubular epithelial cells showed low expression of further study. SN12C contains a wild-type allele of VHL PTTG1. In contrast, 7 of 9 (78%) ccRCC cell lines examined (VHL wild-type) whereas 786-O contains a mutated allele have easily detectable levels of PTTG1. Notably, one of the 2 ofVHL(VHLnull). cell lines that did not show high levels of PTTG1 (SW-156) is To establish a functional role for PTTG1 in ccRCC, the not tumorigenic in vivo (data not shown). Two PTTG1- endogenously expressed oncogene was targeted using RNA

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sion was diminished by siRNA transfection. The efﬁciency of A B siRNA-mediated PTTG1 knockdown was analyzed by both immunoblotting and immunoﬂuorescence, which revealed almost complete ablation of PTTG1 (Supplementary Fig. S2). Subsequent gene expression microarray analysis indicated that expression of several genes was affected by PTTG1 downregulation, including the Rho GTPase-activating proto-oncogene ECT2 (Supplementary Table S3). ECT2 is a guanine exchange factor (GEF) for the Rho family of GTPase's (Rho, Rac, CDC42), and is capable of activating these proteins. Activation of Rho and subsequent Rho- CD3 dependent actin polymerization is critical for regulating actin dynamics in both cellular locomotion and cellular 2 division (cytokinesis). PTTG1 suppression led to an approx-

Score imately 2-fold decrease in ECT2 signal as measured by gene 1 expression microarray and we confirmed this finding at both the mRNA and protein levels by quantitative RT-PCR (qRT- 0 Normal Low High PCR) and immunoblotting. siRNA inhibition of PTTG1 kidney grade grade ccRCC ccRCC resulted in an approximate 4-fold decrease in ECT2 tran- (n = 3) (n = 8) (n = 8) script in 786-O and Caki-1 ccRCC cells as measured by qRT- PCR (Fig. 5A). This PTTG1-mediated regulation of ECT2 expression was seen at the protein level in 4 cell lines tested Figure 2. PTTG1 protein expression is associated with high-grade (786-O, Caki-1, ACHN, and SN12C; Fig. 5B and C). This ccRCC. PTTG1 protein levels were examined in ccRCC and normal kidney tissue sections by immunohistochemistry. Representative relationship between PTTG1 and ECT2 is also supported nondiseased kidney (A); low-grade ccRCC (B); and high-grade ccRCC (C) by expression levels in ccRCC tumors. Similar to PTTG1, tumors are shown. D, semiquantitative scoring of the image intensity of expression of ECT2 is associated with high Fuhrman grade nondiseased (normal kidney), low-grade (Fuhrman grades 1 and 2), and and poor survival (Fig. 5D and E). In addition, there is a high-grade (Fuhrman grades 3 and 4) ccRCC. , significantly increased scores (P < 0.05) as determined using a Wilcox rank sum test. strong association between PTTG1 and ECT2 expression (Fig.5FandG).Thisstatisticallysignificant (P < 0.001) clinical correlation strengthens the direct relationship interference in SN12C and 786-O cells. Stable knockdowns observed between the two genes at both transcript and were established in these cell lines by lentiviral transduction protein levels in vitro. of shRNA sequences targeting PTTG1. The knockdown effi- PTTG1-mediated upregulation of the ECT2 proto-oncogene ciency over multiple passages was verified by immunoblot- and MMP1 metalloproteinase, 2 genes involved in cellular ting (Fig. 3b). PTTG1 knockdown cells were then tested for motility and invasion, suggested that PTTG1 may play a role their proliferative and colony-formation abilities relative to in promoting the cellular locomotion/invasion phenotype as both the parental line and a vector-transduced control cell opposed to a proliferative phenotype (Fig. 3C). To examine the line. There was only a marginal effect on the proliferative role PTTG1 function has on this aspect of ccRCC cell behavior, ability of 786-O cells and no significant effect on SN12C cells we compared the invasive ability of several ccRCC cell lines in vitro in standard monolayer liquid culture as measured by before and after PTTG1 knockdown. Matrigel-coated Boyden MTS assays (Fig. 3C). Cells were cultured in soft agar to chamber invasion assays showed that siRNA-mediated deple- assess the colonigenic potential of nonadherent cells. Colony tion of PTTG1 could reduce the invasive potential of all 4 cell formation in the knockdown cells was diminished consistent lines tested when compared with transfection with a nontar- with a role for the PTTG1 oncogene in ccRCC tumor geting siRNA (P < 0.05; Fig. 6). initiation and early growth (Fig. 3D and E). The importance Due to the reduction of ECT2 expression following PTTG1 of PTTG1 in ccRCC tumorigenesis was further shown by the knockdown, we also investigated the effects of directly target- decrease in tumor growth observed in nude mice upon ing the ECT2 oncogene in ccRCC cells. Moreover, ECT2 knock- targeting the oncogene in ccRCC cell lines. When subcuta- down in HK-2 cells rescued the effect of overexpression of neously xenografted into nude mice, both targeted lines PTTG1 on both cell proliferation and colony formation (Fig. 7). showed significantly reduced tumorigenicity relative to However, in contrast to PTTG1, siRNA inhibition of ECT2 parental and control cells (Fig. 4). Mean tumor volume was expression resulted in a dose-dependent growth inhibition of reduced by approximately 50% in both the 786-O-shPTTG1 ccRCC cells that promptly acquire a spindle-shaped morphol- and SN12C-shPTTG1 lines. ogy (Supplementary Fig. S1). Flow-cytometric analysis revealed that cells lacking ECT2 accumulate in G2/M phase, consistent Identification of PTTG1-associated genes with ECT2 role in cytokinesis. Subsequent immunofluorescent To identify potential proteins that are influenced by staining showed frequent multinucleation resulting from a PTTG1, we carried out gene expression analysis on mock- failure to complete cytokinesis (Supplementary Fig. S3), as transfected cell lines and cell lines in which PTTG1 expres- previously described in glioma cells (43).

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A Normal VHL wild-type VHL null

HUVEC HK2 SN12C Coki-1 ACHN RXF393 UO31 TK10 A498 SW155 786-O

PTTG1

β-Actin

786-O ParentalVector controlshPTTG1 SN12C ParentalVector control shPTTG1 PTTG1

β-Actin

C 5 7 SN12C 786-O

3 Figure 3. Knockdown of PTTG1 prevents colony formation in soft agar. PTTG1 protein levels were 3 determined by Western blotting in the indicated cell lines (A) and cell lines stably transduced to express growth Fold-change control or shRNA sequences targeting PTTG1 (B). C, cell number was measured using 1 1 an MTS assay in 786-O cells and SN12C cells 0 Hours 60 120 0 50 100 transfected with control or PTTG1-targeting siRNA. D and E, representative images (D) and quantiﬁcation Parental Negative control siRNA PTTG1 siRNA (E) of 786-O and SN12C cells and cells transfected D with control or PTTG1-targeting shRNAs after culture Parental Vector shPTTG1 in soft agar for 3 weeks (, P < 0.01). 786-O

SN12C

E Parental 250 Vector control shPTTG1

150

Colonies/field **

50 **

SN12C 786-O

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PTTG1, underscoring the importance of this oncogene in A ccRCC pathogenesis (data not shown). PTTG1 overexpression in human tumor samples is corre- 2,000 786-O Parental lated with high Fuhrman grade and high tumor stage,

Vector Control ) 3 AB PTTG1 shRNA 1,000

ECT2 Tumor volume (mm volume Tumor PTTG1

0 β-Actin 0 Days after implition 30 60 B C Caki-1 ACHN SN12C 2,000 PTTG1 siRNA: – + – + – + Control siRNA: + – + – + – SN12C Parental ECT2

PTTG1 Vector Control

) β-Actin 3

PTTG1 shRNA DE 1,000 800 1.0 500 0.8 Low, n = 132 400 0.6 300 ECT2 expression ECT2 0.4 P = 3.77e –06 200 Tumor volume (mm volume Tumor

100 0.2 High, n = 47

0 Cancer specific survival 0.0 0 Grade: N 1 2 3 4 0 10 20 # Sample: 12 17 62 57 29 Years 0 Days after implition 30 60

F G PTTG1 ECT2

Figure 4. Knockdown of PTTG1 reduces the growth of tumor cell 800 R = 0.7 xenographs. Tumor volume of 786-0 and SN12C cells transfected with P < 0.001 2,000 control or PTTG1-targeting shRNAs. 600 1,500

400 ECT2 1,000

200 Discussion 500

Although copy number gain of chromosome 5q constitutes 0 0 500 1,000 1,500 2,000 2,500 0 N 1 2 3 4 the second most frequent chromosomal aberration in ccRCC, PTTG1 Grade the significance of this abnormality in the development and progression of the disease has not been explained to date. We show that PTTG1, a recently described oncogene, is located Figure 5. PTTG1 expression influences ECT2 expression. Gene in this region of amplification. PTTG1 is both overexpressed expression microarray analysis was carried out on control and PTTG1 targeting siRNAs in 786-O and Caki-1 cells. A, ECT2 expression in 786-0 in poor prognosis renal tumors and shown to play an impor- and Caki-1 cells and cells transfected with control or PTTG1-targeting tant functional role in vitro. Following PTTG1 knockdown, the siRNAs. B, ECT2 proteins levels as determined by Western blotting in effect on ccRCC cell proliferation is modest; however, ccRCC 786-O cells and cells transfected with control or various PTTG1-targeting cells exhibit renewed anchorage dependence. As growth in siRNAs. C, PTTG1 and ECT2 expression in other indicated ccRCC cell lines following transfection with either control or PTTG1-targeting siRNA. soft agar is a hallmark of neoplastic transformation, it seems D, Expression levels of ECT2 in human ccRCC tumor samples. that efficient targeting of the endogenously overexpressed Nondiseased specimens (N) and ccRCC tumors are shown with the PTTG1 oncogene can cause ccRCC cells to revert to a non- tumors grouped by grade (1–4). The number of samples in each group is transformed phenotype. When these cells are injected into shown. Increased ECT2 expression is associated with elevated nuclear P < – mice, they show a significant reduction in tumorigenesis. The grade ( 0.01). E, Kaplan Meier plot of PTTG1 expression and cancer- specific survival in ccRCC samples partitioned into high and low groups tumors that eventually develop in these mice show restored as described in Materials and Methods. F and G, correlation between PTTG1 expression, indicative of selection for cells expressing PTTG1 and ECT2 expression levels in clinical ccRCC samples.

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PTTG1 and Aggressive Renal Cancer

indicative of more aggressive tumors and more invasive AB tumors, respectively. This clinical observation was corrobo- rated by cellular studies in vitro, which showed PTTG1-dependent overexpression of 2 important activators of invasion, the MMP1 metalloproteinase and the ECT2 Rho-GTPase GEF, a proto-oncogene discovered based upon its transforming abilities. Furthermore, suppression of PTTG1 significantly reduces the invasive phenotype of these cells in vitro. Due to its overexpression in high grade, high stage, and poor prognosis tumors, as well as, clinical correlation to PTTG1 expression, we examined the relationship between PTTG1 and ECT2 C D expression. fl 200 Control siRNA PTTG1 siRNA At the protein level, ECT2 expression is strongly in u- ** ** ** * enced by PTTG1 expression in ccRCC cells in vitro,and these genes are coordinately overexpressed in a subset of high stage, poor prognosis ccRCC tumors clinically. It is 100 interesting to note that Ito and colleagues identified a number of other Rho family members and Rho-interacting proteins as probable PTTG1 downstream targets in ESCC Cells/field 0 (22). However, whether PTTG1 lies upstream, downstream, 786-O Caki-1 ACHN SN12C or part of a complex with ECT2 remains to be examined in greater detail. The association between ECT2 and PTTG1 Figure 6. PTTG1 knockdown reduces the invasive ability of ccRCC cell implies that PTTG1 promotion of the invasive/anchorage lines. Representative images of crystal violet-stained parental 786-O independent growth contributes more to the tumorigenic cells (A) and cells transfected with control (B) or PTTG1-targeting (C) phenotype than an effect on cellular proliferation. Hirata siRNAs following chemotactic invasion through Matrigel. D, quantification of invasive cells in indicated cell lines transfected with and colleagues implicated ECT2 in esophageal carcinoma control or PTTG1 targeting siRNAs. (, P < 0.001, , P < 0.05). invasion, suggesting that the PTTG1 and ECT2 oncogenes

A Control shRNA ECT2 shRNA B Vector : + – + – PTTG1: – + – +

PTTG1 ** ECT2

β-Actin

C Vector PTTG1 D **

Control shRNA

ECT2 shRNA

Figure 7. ECT2 knockdown prevents PTTG1-medidated growth. HK-2 cells that stably overexpressed PTTG1 were treated with ECT2-targeted shRNA (A). PTTG1 and ECT2 protein levels were determined by Western blotting in the indicated cell. B, cell number was measured using an MTS assay (, P < 0.01). C, colonies formation of indicated modiﬁed HK-2 cell lines after culture in soft agar for 5 weeks. D, quantiﬁcation of colonies number for the indicated cell lines (, P < 0.01).

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Wondergem et al.

may be coordinately activated in ESCC much like we have Development of methodology: B. Wondergem, Z. Zhang, D. Huang, A. Ooi, B. T. Teh observed in ccRCC (44). Analysis and interpretation of data (e.g., statistical analysis, biostatistics, In the ongoing search for molecular points of therapeutic computational analysis): B. Wondergem, Z. Zhang, J. Koeman, D. V. Hof, K. A. intervention for various malignancies, transcriptional activa- Furge, B. T. Teh Writing, review, and/or revision of the manuscript: B. Wondergem, tors such as PTTG1 have generally not been successful as drug Z. Zhang, D. Huang, C. K. Ong, B. Lane, K. A. Furge, B. T. Teh targets, though this has been a point of contention (45). In Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J. Anema, R. J. Kahnoski, B. T. Teh addition to the targeted inhibition of kinases, it has previously Administrative, technical, or material support (i.e., reporting or orga- been shown that GEFs, such as ECT2, represent viable targets nizing data, constructing databases): B. Wondergem, Z. Zhang, D. Petillo, for the development of novel antiinvasion cancer therapeutics J. Anema, B. Tean Teh Study supervision: B. Wondergem, Z. Zhang, B. Lane, K. A. Furge, B. T. Teh (46). The development of resistance to monotherapy and fi incomplete response rates has necessitated the identi cation Acknowledgments of additional targets for anticancer drug development for use in This work is dedicated to the memories of William (Bill) Wondergem and his either personalized therapeutic regimens or combination ther- grandfather Casey Wondergem. Casey Wondergem was a founding member of fi the Van Andel Institute executive committee. His grandson, Bill, was working in apies. Herein, we have identi ed PTTG1 as a potential candi- our research lab while applying to medical school. They will both be greatly date oncogene in ccRCC, contributing to both tumorigenesis missed. The corresponding authors gratefully acknowledge the Van Andel and the invasive phenotype of ccRCC cells, and furthermore, Research Institute and The Gerber Foundation for their support. We thank the Cooperative Human Tissue Network of the National Cancer Institute for identified a downstream effector of this oncogene in the Rho- providing the renal tumor cases. We also thank Sabrina Noyes for assistance GEF ECT2. in preparation and submission of the manuscript. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked Disclosure of Potential Conflicts of Interest advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this No potential conflicts of interest were disclosed. fact.

Authors' Contributions Received August 11, 2011; revised June 4, 2012; accepted June 6, 2012; Conception and design: B. Wondergem, Z. Zhang, D. Huang, B. T. Teh published OnlineFirst July 17, 2012.

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Expression of the PTTG1 Oncogene Is Associated with Aggressive Clear Cell Renal Cell Carcinoma

Bill Wondergem, Zhongfa Zhang, Dachuan Huang, et al.

Cancer Res 2012;72:4361-4371. Published OnlineFirst July 17, 2012.

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