KLF6 Suppresses Metastasis of Clear Cell Renal Cell

Home , KLF6

Published OnlineFirst October 25, 2016; DOI: 10.1158/0008-5472.CAN-16-0348 Cancer Molecular and Cellular Pathobiology Research

KLF6 Suppresses Metastasis of Clear Cell Renal Cell Carcinoma via Transcriptional Repression of E2F1 Yu Gao1,2, Hongzhao Li1,2, Xin Ma1,2, Yang Fan1,2, Dong Ni1,2, Yu Zhang1,2, Qingbo Huang1,2, Kan Liu1,2, Xintao Li1,2, Lei Wang1,2, Liangyou Gu1,2, Yuanxin Yao1,2, Qing Ai1,2, Qingshan Du1,2, Erlin Song3, and Xu Zhang1,2

Abstract

The transcription factor KLF6 has an essential role in the silencing reversed these properties. These effects were corrob- development and metastasis of multiple human cancers. Para- orated in a metastatic model system, where we observed a doxically, KLF6 expression was found to be attenuated in pri- greater number of pulmonary metastatic lesions formed by mary metastatic clear cell renal cell carcinoma (ccRCC), such ccRCC cells where KLF6 was silenced and E2F1 enforced. that it is unclear how KLF6 affects malignant progression in this Analysis of clinical specimens of ccRCC revealed that low levels setting. In this study, we demonstrate that KLF6 attenuation in of KLF6 and high levels of E2F1 correlated closely with ccRCC renal cells is sufficient to promote E2F1-mediated epithelial– development. Overall, our results established the significance mesenchymal transition and metastatic prowess. In a mouse of activating the KLF6–E2F1 axis in aggressive ccRCC, defining a xenograft model of human ccRCC, silencing KLF6 increased novel critical signaling mechanism that drives human ccRCC tumor cell proliferation and malignant character, whereas E2F1 invasion and metastasis. Cancer Res; 77(2); 1–13. 2016 AACR.

Introduction The Kruppel-like€ factor 6 (KLF6) gene is a tumor suppressor that is deregulated and inactivated by gene loss or somatic Renal cell carcinoma (RCC) is the second leading cause of death mutation in various cancers, such as colorectal cancer (6), in patients with urological malignant tumors and accounts for prostate cancer (7), and glioblastoma (8). Our previous tissue 4.2% of all adult malignancies (1). Clear cell RCC (ccRCC) is the profiling results indicated that KLF6 was significantly down- most commonly observed of the 5 major subtypes of RCCs: regulated in metastatic specimens. Gene set enrichment anal- the clear cell, papillary, chromophobe, collecting duct, and the ysis (GSEA) suggested that the aberrant expression of E2F1 is a unclassified types. Metastasis occurs in the later stages and predicts key regulator of the entire metastatic process. Bioinformatics very poor clinical outcome (2). The 5-year survival rate of met- predicted the strong linkage between KLF6 attenuation and astatic RCC is only 10% because of resistance to chemotherapy E2F1 activation in metastatic processes. E2F1 plays an impor- and radiation therapy (3). Cytokine therapy with IFNa and IL2 tant role in regulating development, differentiation, prolifera- was introduced in the past decades, but this approach does not tion, cellular signal transduction, and apoptosis (9–12). Cer- function well in late-stage disease management (4). Molecular tain studies reported that E2F1 disrupts a range of pathways in targeting drugs, such as sunitinib and temsirolimus, have been numerous cancers and is closely correlated with clinical para- recently approved and widely used for advanced metastatic meters (13–15). We had also previously identified that E2F1 is ccRCC. However, the therapeutic effects of these drugs are limited upregulated in ccRCC progression (16). to a short interval; overall survival remains poor (5). Thus, the In this study, we illustrated that KLF6 downregulation promot- characterization of genetic alterations underlying the key steps in ed epithelial–mesenchymal transition (EMT) and metastatic the metastatic progression of ccRCC is crucial. transformation. On the basis of bioinformatics analysis, the present work focused on the combined effects of KLF6 and E2F1 in the migration and invasion of ccRCC cells in vitro and 1Department of Urology, Chinese PLA General Hospital/Chinese PLA Medical in vivo, as well as their mechanic relationship and clinical rele- School, Beijing, PR China. 2State Key Laboratory of Kidney Diseases, Chinese PLA General Hospital/Chinese PLA Medical School, Beijing, PR China. 3Depart- vance. A cooperative requirement between KLF6 and E2F1 in ment of Urology, The First Affiliated Hospital of Harbin Medical University, metastasis was suggested by the stimulation of EMT-related Harbin, China. process, thereby causing enhanced tumor progression and distant fi Note: Supplementary data for this article are available at Cancer Research organ colonization. Therefore, the functional signi cance of the Online (http://cancerres.aacrjournals.org/). KLF6 and E2F1 in the development and metastasis of ccRCC was Y. Gao, H. Li, and X. Ma contributed equally to this article. investigated. Corresponding Author: Xu Zhang, Department of Urology, Chinese PLA General Hospital, 28, Fuxing Road, Haidian District, Beijing 100853, PR China. Phone: 86- Materials and Methods 10-66938008; Fax: 86-10-68223575; E-mail: [email protected] Ethics statement doi: 10.1158/0008-5472.CAN-16-0348 Written informed consent was obtained from each individual 2016 American Association for Cancer Research. who underwent nephrectomy prior to sample collection. The

www.aacrjournals.org OF1

Gao et al.

study was approved by the Protection of Human Subjects Com- scores (intensity score percentage score), scores less than 2 were mittee of the Chinese People's Liberation Army (PLA) General considered as negative staining, 2–3 indicated weak staining, 4–6 Hospital. All animal studies were conducted according to the was moderate staining, and >6 was strong staining (18). For the in guidelines of the Institutional Animal Care and Use Committee of vivo angiogenesis experiments, staining with CD34 was quantified the Chinese PLA General Hospital. on the basis of the intensity of microvessel staining in selected fields. The primary antibody sources are listed in Supplementary Patients and clinical materials Table S1. The study cohort included 114 patients with ccRCC admitted to the Urology Department of the Chinese People's Liberation Army Cell line and cell culture (PLA) General Hospital from January 2005 to December 2010. The 786-O, OS-RC-2, 769-P, and Caki-2 human ccRCC cell Cancerous tissues were collected and immunohistochemically lines and human umbilical vein endothelial cells (HUVEC) were analyzed. All tissue samples were clinically and pathologically acquired from the National Platform of Experimental Cell fi fi con rmed to be of the clear cell type and classi ed according to the Resources for Sci-Tech. SN12-PM6 cells were preserved in our fi 2011 Union for International Cancer Control TNM classi cation laboratory, and all cell lines were obtained in 2015 and authen- of malignant tumors. The nuclear grade was determined with the ticated in September 2015 by the short tandem repeat analysis Fuhrman nuclear grading system. method. 786-O, OS-RC-2, 769-P, Caki-2, and SN12-PM6 cells were cultured in DMEM (HyClone), McCoy 5A medium RNA isolation and quantitative real-time PCR (HyClone), DMEM/F12 (HyClone), and high glucose-DMEM Total RNA was extracted with the TRIzol reagent (ComWin with 10% FBS (Gibco), respectively. Biotech). cDNA synthesis of regular genes was performed with a TransScript First-Strand cDNA Synthesis SuperMix Kit (TransGen Biotech). Afterward, quantitative real-time PCR (qRT-PCR) was Tube formation assay – performed with qPCR SYBR Green SuperMix (TransGen Biotech). Growth factor reduced basement membrane extract (BME; Relative mRNA expressions were normalized to peptidylprolyl Trevigen) was added to each well of a 96-well plate and prepared DDC isomerase A (PPIA) with the 2 T method. The primer for 1 hour at 37 C. HUVECs cocultured with ccRCC cells from sequences used are listed in Supplementary Table S1. different groups for 48 hours were pre-incubated with serum-free DMEM for 1 hour and seeded into the BME-coated wells. Methylation-specific PCR Genomic DNA from ccRCC tissues and cell lines was modified Western blot assay by sodium bisulfite treatment as previously described (17). CpG Western blot assays were performed as previously described island spanning the KLF6 gene was found (Supplementary Fig. (19). The information on all primary antibodies is listed in S1). Methylation-specific PCR (MSP) primers for KLF6 were Supplementary Table S1. designed and synthesized (Invitrogen). For detecting the methylated DNA (M) and unmethylated DNA (U), the primers were designed and the sequences used are listed in Supplementary MTS assay Table S1. The cells in different groups were seeded into 96-well plates (1,000 cells per well). Absorbance was measured at 24, 48, 72, and 96 hours after seeding. All experiments were performed triplicate. Gene expression profile and GSEA analysis Our previous study investigated the genes that promoted ccRCC metastasis in 4 primary metastatic and 5 nonmetastatic Cell migration and invasion assay tumor samples. The U133 plus 2.0 Array was used to identify the Twenty-four–well plates were applied with Boyden chambers differently expressed genes between the primary metastatic and containing Transwell membrane filter (8 mm). For the invasion nonmetastatic ccRCC samples. The microarray data were depos- assay, diluted Matrigel (BD Biosciences) was used. The migration ited with the Gene Expression Omnibus (GEO) as a microarray and invasion assays were performed as previously described (20). dataset (GSE47352). The dataset is publicly available and acces- sible through the NCBI GEO website. A heatmap was generated with part of the clustered genes on the data set (Supplementary Wound-healing assay Fig. S2). GSEA was applied to run the data for the microarray Images of wounds were captured at 0 and 12 hours after m according to the manufacturer's protocol. The C2tft (transcript scratching with a sterile 200 L pipette. The coverage of the factor target) calculation was applied and the top factors were scratched area was measured at three different positions. All normalized and conducted. experiments were performed in triplicates.

Immunohistochemistry Immunofluorescence Immunohistochemistry (IHC) was performed on renal cancer Cells of different groups were seeded and grown on glass slides samples and xenograft tumors. As previously described, staining at 24 hours prior to the proper experiment. After fixation with 4% intensity was scored 0 (negative), 1 (low), 2 (moderate), and 3 paraformaldehyde/PBS for 15 minutes, cells were washed once (high). Staining range was scored 0 (0% stained), 1 (1%–25% with PBS and permeabilized with 0.5% Triton X-100. Coverslips stained), 2 (26%–50% stained), and 3 (51%–100% stained). The were stained with the primary antibodies (KLF6 and E2F1) at final score was obtained by multiplying the intensity scores with 37C for 1 hour, respectively. Nuclei were stained by application staining range, and the results ranged from 0 to 9. For the final of 0.2 mg/mL DAPI.

OF2 Cancer Res; 77(2) January 15, 2017 Cancer Research

KLF6 Transcriptionally Represses E2F1 in ccRCC Metastasis

siRNA and plasmid constructs Statistical analysis siRNAs against KLF6 and E2F1 were designed (Supplementary Statistical analysis was performed with SPSS 18.0 (SPSS Inc.). Table S1) and synthesized by GenePharma. A fragment of the Normally distributed data were expressed as mean SD, and KLF6 coding sequence was inserted into the lentiviral vector pLV- comparisons were performed with Student t test. Categorical data EGFP-(2A)Puro (Inovogen Tech. Co). XbaI and EcoRI were used to were analyzed with either Fisher exact test or the c2 test. Correla- generate pLV-EGFP-KLF6. Lentivirus-encoding DNA was pack- tions of gene expression were determined with the Pearson aged as previously described (21). For knockdown of KLF6 and coefficient. Kaplan–Meier and log-rank tests were used for the E2F1, siKLF6#2 and siE2F1 sequences were cloned into the overall survival analysis. pLVshRNA-EGFP-(2A)Puro vector. The primer sequences are listed in Supplementary Table S1. Extensions containing 2 restric- tion sites for KpnI and HindIII were designed in each primer to Results facilitate the cloning of this fragment into the pGL3-basic lucif- Downregulation of KLF6 in primary metastatic ccRCC tissues erase vector (Promega). To investigate the potential mechanism that promotes distant metastasis of ccRCC, mRNA expression profiling was performed with 4 primary metastatic and 5 primary nonmetastatic ccRCC Bioinformatics of the E2F1 promoter tissue samples. The tissue profiles indicated that KLF6 mRNA The human promoter sequence of E2F1 was retrieved from levels are significantly reduced in primary metastatic ccRCC as NCBI and defined as a putative promoter located in a 700-bp compared with primary nonmetastatic ccRCC (Fig. 1A). Subse- region spanning 700 to þ1 bp relative to the transcriptional start quent validation was performed on the differential gene expres- site. The sequence was amplified from cell line 293T. Genomatix sion between the two groups. Freshly collected specimens were (www.genomatix.de/en/index.html) was used to identify tran- tested after matching according to age and gender. Similar to the scription factor–binding sites for KLF6 within the human E2F1 profiling data, the mRNA and protein expression levels of KLF6 promoter. The mutation construct was created by Genewiz. The also significantly declined upon ccRCC metastasis (Fig. 1B and C). mutation was confirmed by sequencing. As expected, KLF2 expression was attenuated in primary metastatic ccRCC. However, KLF9 expression was not consistent with Luciferase assay the profiling results (Fig. 1C). Furthermore, IHC illustrated that 293T cells were plated at a density of 5 105 cells per well in 6- the KLF6 protein was mainly localized in the nuclei of cancer cells well plates prior to transfection. Transfection started when cell from primary metastatic and primary nonmetastatic samples. As density reached approximately 60% confluency. The pGL3-E2F1 shown in Supplementary Table S2, a total of 114 ccRCC cases were vector, a pRL construct containing the Renilla luciferase reporter, analyzed. The nonmetastatic group included 79 cases, and the and a pcDNA3.1 control or increasing doses (0.5, 1, and 2 mg) of metastatic group contained 35 cases. In the nonmetastatic group, the pcDNA3.1-KLF6 expression vector were cotransfected into absent/weak immunostaining was observed in 33% (26 of 79) of 293 cells. After 48 hours, luciferase activity was measured with the the tumors, moderate staining in 29% (23 of 79) of the tumors, Dual Luciferase Reporter Assay System (Promega). The value of and strong staining in 38% (30 of 79) of the tumors. In compar- firefly luciferase activity was normalized to that of the Renilla ison, absent/weak immunostaining was observed in 69% (24 of activity. 35) of the tumors, moderate staining in 17% (6 of 35) of the tumors, and strong staining in 14% (5 of 35) of the tumors. KLF6 expression was significantly decreased in primary tumors when Chromatin immunoprecipitation metastasis occurred. Chromatin immunoprecipitation (ChIP) assays were per- In 12 pairs of comparative tissues, complete and partial meth- formed on 786-O and Caki-2 cells with an antibody for KLF6, ylation of the KLF6 promoter region were found in metastatic with anti-rabbit IgG as the negative control. The interaction with samples, whereas in 4 cells lines, the methylation status was also predicted promoters was detected with qRT-PCR. The detailed evaluated (Fig. 1E). primer sequences are shown in Supplementary Table S1. The To investigate the correlation of KLF6 expression levels with whole procedure was performed according to the protocol pro- ccRCC metastasis, the clinicopathologic parameters and KLF6 vided by the ChIP Assay Kit (Upstate EZ-CHIP, 17-371). Lysates mRNA levels with metastatic incidence in 87 ccRCC tissue speci- were immunoprecipitated with an antibody specific for KLF6, mens were analyzed with univariate and multivariable logistic normal rabbit IgG, and a positive control. regression models. Patient characteristics are provided in Supple- mentary Table S3. We ranked the relative KLF6 mRNA expression In vivo orthotopic xenograft tumor and metastatic model in sequence and selected the median (0.2799) of the entire dataset The cells (1 106 in 0.1 mL of sterilized PBS) in respective as the cutoff point between the high KLF6 group and the low KLF6 groups were injected into the left kidneys of 4- to 5-week-old male group (Fig. 1F). Univariate analysis indicated that a higher clinical nude mice (10 mice per group). Lesions, including the orthotopic T stage [OR, 6.034; 95% confidence interval (CI), 1.912–19.047; P xenograft tumors and lung metastatic nodules, were monitored in ¼ 0.002; Table 1) and lower KLF6 expression (OR, 6.308; 95% CI, vivo with a molecular imaging system (NightOWL II LB983; 1.898–20.959; P ¼ 0.003; Table 1) were closely associated with ref. 22). The signal intensity of luc-labeled cells from lung tissues developing metastasis. Multivariate analysis demonstrated that a represented the amount of lung metastatic lesions. For the met- higher clinical T stage (OR, 6.359; 95% CI, 1.782–22.690; P ¼ astatic seeding model, approximately 5 105 786-O cells were 0.004) and lower KLF6 expression (OR, 6.581; 95% CI, 1.829– injected into the tail vein of each nude mouse. For SN12-PM6 23.675; P ¼ 0.004) were correlated with the ccRCC progression established model (6 mice per group), 3 months later, the images related to other clinicopathologic parameters, including gender, were taken. age, body mass index (BMI), and the Fuhrman grade (Table 1).

www.aacrjournals.org Cancer Res; 77(2) January 15, 2017 OF3

Gao et al.

Figure 1. Downregulation of KLF6 in primary metastatic ccRCC tissues. A, Heatmap illustration of KLF6, KLF2, and KLF9 transcript abundance determined by tissue microarrays between the primary metastatic and nonmetastatic ccRCC groups. Blue shading, low; red shading, high expression of mRNA. B, Protein expressions of KLF6 in clinical samples was determined by Western blot assay in patients with primary metastatic and nonmetastatic ccRCC. C, Differentiated mRNA levels of KLF6, KLF2, and KLF9 in primary metastatic and nonmetastatic ccRCC tissues. D, ccRCC tissues were elucidated by KLF6 antibody, being classiﬁed into the absent/ weak, moderate, and strong groups. KLF6 protein exhibited distinguished expressions between primary metastatic and nonmetastatic ccRCC (P ¼ 0.002). E, Representative MSP results in ccRCC tissues and cell lines. IVD, in vitro methylated DNA; M, methylated alleles; NL, normal blood lymphocyte DNA; U, unmethylated alleles, F, Of the enrolled 87 cases, the relative mRNA levels were tested in convenience of the logistic regression analysis. , P < 0.05; , P < 0.01; , P < 0.001.

OF4 Cancer Res; 77(2) January 15, 2017 Cancer Research

KLF6 Transcriptionally Represses E2F1 in ccRCC Metastasis

Table 1. Univariate and multivariable logistic regression models comparing patients developing metastatic disease with patients free from metastatic incidence Univariate analysis Multivariate analysis Variables OR (95% CI) P OR (95% CI) P Gender Male 1 Female 1.684 (0.595–4.767) 0.326 Age, y <60 1 60 0.798 (0.282–2.258) 0.67 BMI, kg/m2 <25 1 25 0.706 (0.250–1.993) 0.510 T stage T1 þ T2 1 1 T3 þ T4 6.034 (1.912–19.047) 0.002 6.359 (1.782–22.690) 0.004 Fuhrman grade 1 þ 21 3 þ 4 0.462 (0.095–2.244) 0.338 KLF6 levels High 1 1 Low 6.308 (1.898–20.959) 0.003 6.581 (1.829–23.675) 0.004 NOTE: TNM staging: TNM stage grouping was assigned according to the 2009 TNM staging classiﬁcation system.

Overall, the results illustrated that KLF6 downregulation is highly a rounded shape with cytoskeletal remodeling. Visualizing cells associated with ccRCC metastasis. with confocal microscopy showed that KLF6 gene silencing in 786-O and Caki-2 cells elevated the expression of N-cadherin Loss of KLF6 promotes ccRCC tumorigenesis in vitro and vimentin but decreased E-cadherin expression. KLF6 over- To corroborate the above-mentioned data derived from human expression in 769-P and OS-RC-2 cells conferred a phenotype ccRCC samples and to validate the function of KLF6 in cancer cells, with the reversed expression of N-cadherin, E-cadherin, and KLF6 expression was measured in a panel of ccRCC cell lines with vimentin. The above results, taken collectively, suggested that different malignant behaviors. As shown in Fig. 2A, KLF6 was KLF6 might suppress EMT of ccRCC cells and thereby inhibit expressed at a relatively high level in 786-O and Caki-2 cells but at metastasis. a relatively low level in 769-P and OS-RC-2 cells. The reduction of KLF6 protein levels was observed in the 786-O and Caki-2 ccRCC E2F1 is aberrantly activated in ccRCC and is a direct cell lines after treatment with 3 siRNA sequences. In addition, the target of KLF6 lentiviral plasmid introduction of KLF6 in OS-RC-2 and 769-P GSEA was used to analyze microarray data (GSE47352). As cells obviously increased the KLF6 expression compared with the previously illustrated, the profiling sheet included 4 primary empty vector (EV; Fig. 2A). metastatic and 5 primary nonmetastatic samples. The C2tft cal- After confirming the knockdown efficacy of siRNA transient culation of gene clusters containing the matched binding motifs transfection by the Western blot assay, the MTS assay was per- (NTTTCGCGCS) was conducted (Fig. 3B and Supplementary Fig. formed and 786-O and Caki-2 cells transfected with siKLF6#2 S2). Results revealed that E2F1 is the top gene signature that was showed remarkably enhanced growth relative to cells in the siNC enriched in differentially expressed genes (Fig. 3A). To gain more group (Fig. 2B). Conversely, KLF6 overexpression in OS-RC-2 and reliable outcomes, we combined several reference parameters, 769-P cells significantly inhibited cell proliferation as compared including the normalized enrichment score (NES), P value, and with the EV group. fold change. The ranking flowchart is shown in Supplementary Transwell with or without a Matrigel coating were applied for Fig. S2. E2F1 was significantly found to be the key upregulated the invasion and migration assays of ccRCC cells. 786-O and factor in the primary metastatic group. This result was consistent Caki-2 cells treated with siKLF6#2 showed enhanced invasive with our previously published findings. The validation of E2F1 abilities compared with cells treated with siNC. The invasive expression in a larger sample size was subsequently conducted by and migratory abilities were both significantly attenuated in qRT-PCR and Western blot analysis (Fig. 3C). The correlation KLF6-infectedOS-RC-2and769-Pcells(Fig.2C).Inthe between the KLF6 and E2F1 mRNA levels in ccRCC tissue samples wound-healing assay, KLF6 knockdown augmented 786-O and (n ¼ 45) was further analyzed by linear regression analysis. As Caki-2 cell migration at 12 hours after scratching; 769-P and shown in Fig. 3D, E2F1 expression was progressively reduced (r2 ¼ OS-RC-2cellstransfectedwithKLF6lentiviralparticlesdis- 0.3546, P < 0.0001) compared with the associated elevation of played decreased migratory capacity compared with the con- KLF6 expression. The 769-P and OS-RC-2 cell lines showed higher trols (Fig. 2D). A series of markers related to the EMT, including E2F1 expression and low levels of KLF6 expression, whereas the N-cadherin, E-cadherin, and vimentin, were detected by immu- 786-O and Caki-2 cell lines showed decreased E2F1 expression nofluorescent staining, thereby suggesting the malignant shift and high levels of KLF6 expression (Fig. 3E). A double-color in marker expression. As shown in Fig. 2E, impaired KLF6 immunofluorescent test showed that endogenous KLF6 and expression in 786-O and Caki-2 cells transformed the cell E2F1 proteins were localized in the nuclei of most 769-P cells. morphology from a rounded, epithelial-like contour to a spin- A merged signal suggested the colocalization of both proteins dle shape. In contrast, the polarized morphology of 769-P and (Fig. 3F). Reduced KLF6 expression was associated with a strong OS-RC-2 cells was disturbed after KLF6 overexpression, causing induction of E2F1 mRNA expression in 786-O cells. Inversely,

www.aacrjournals.org Cancer Res; 77(2) January 15, 2017 OF5

Gao et al.

Figure 2. KLF6 is a tumor suppressor in ccRCC. A, Relative KLF6 protein levels in different ccRCC cell lines (left) and the efficiency of knockdown and overexpression were validated in related cell lines (right). B, MTS assay showed that KLF6 knockdown promoted the tumor growth in 786-O and Caki-2 cells, whereas KLF6 overexpression in OS-RC-2 and 769-P reduced the proliferation velocity. C, KLF6 attenuation significantly increased migration and invasion in 786-O and Caki-2. On the contrary, KLF6 overexpression suppressed tumor aggressiveness in vitro in OS-RC-2 and 769-P (P < 0.001). D, For wound-healing assay, in 786-O and Caki-2 cells, KLF6 knockdown significantly increased the number of viable cells (P < 0.001); in 769-P and OS-RC-2 cells, KLF6 introduction inhibited the cell viability (P < 0.001). E, Immunofluorescent staining was used to explore the EMT-related protein alteration after KLF6 knockdown or upregulation in different cell lines. Magnification, 600. , P < 0.05; , P < 0.01; , P < 0.001.

OF6 Cancer Res; 77(2) January 15, 2017 Cancer Research

KLF6 Transcriptionally Represses E2F1 in ccRCC Metastasis

www.aacrjournals.org Cancer Res; 77(2) January 15, 2017 OF7

Gao et al.

KLF6 was overexpressed in 769-P cells. The expression of E2F1 duction of anti-E2F1 plasmids, the cells with elevated invasion mRNA was obviously suppressed upon KLF6 plasmid transfection suppressed N-cadherin, ZEB1, and vimentin expression. There- (Fig. 3G). fore, KLF6 mitigation significantly stimulated E2F1 expression E2F1 is overexpressed in ccRCC; thus, its upstream regulator and EMT-related gene expression levels (Fig. 4B). was explored. The sequence upstream of the transcriptional The morphologic changes in different cell lines were observed. initiation site by Genomatix (www.genomatix.de/en/index. Compared with the shNC group cells, stable cells with KLF6 html) was found to contain putative KLF6-binding sites knockdown displayed a spindle-like, fibroblastic morphology, at 485/468, 421/404, 291/272, 230/212, and whereas shE2F1 employment can partially reverse the EMT pro- 176/114. The putative binding sequences are listed in the cess (Fig. 4C). box on the right, with the core element labeled in red (Fig. Angiogenesis is important in malignancy and metastasis; 3H). To determine whether endogenous KLF6 binds to the thus, the functional consequence of KLF6-mediated regulation E2F1 promoter region in vivo, ChIP assays were performed on on E2F1 expression was explored by testing the function of 786-O and Caki-2 cell lysates. Three primers were designed to HUVECs. KLF6 knockdown in Caki-2 increased the expression capture the precipitated DNA fragment. A DNA fragment of E2F1 and boosted the formation of endothelial tubule–like containing the KLF6-binding site (485/468) was signifi- structures. This effect was completely abolished by shE2F1 cantly enriched in chromatin precipitated with an anti-KLF6 transfection, thereby demonstrating that tubule formation is antibody, but no bands were evident in the immunoprecipi- mediated by E2F1 (Fig. 4D, left). In contrast, KLF6 overexpres- tates of the other possible binding segments (Fig. 3I). Our sion in OS-RC-2 cells considerably reduced tubule formation findings implied that KLF6 may directly interact with E2F1 by (Fig. 4D, right). Taken together, these data indicated that ccRCC binding with the promoter. Given the relatively low transient progression was promoted by a key signaling axis involving transfection efficiency in renal cancer cell lines, 293T cells KLF6-mediated E2F1 regulation. were used to perform a luciferase assay. When transfected into 293T cells, the dose-dependent luciferase activity of the pGL3- KLF6 downregulation promotes E2F1-mediated cell E2F1 reporter treated with pcDNA3.1-empty or pcDNA3.1- proliferation and metastasis in vivo KLF6(0.5,1,and2mg) was significantly attenuated compared To investigate the comprehensive function of KLF6 and E2F1 with that of pGL3-basic (Fig. 3J). The activity was also in vivo, 786-O cells of different groups were subsequently measured for a luciferase construct where the core binding injected into the left kidney of a nude mouse. At 8 weeks after element, GGTG (485/468), was replaced with CCAC. The injection, bioluminescent signals in the kidneys were signifi- MUT reporter activity was markedly reversed in 293T cells, cantly higher in the shKLF6 group than in the shNC group; the thereby suggesting that KLF6 downregulates E2F1 expression effect of KLF6 was further mitigated by E2F1 knockdown (Fig. by binding to the site (485/468) in the E2F1 promoter 5A).Allmiceweresacrificed at 8 weeks after injection, and the region (Fig. 3K). kidneys were harvested. Orthotopic implantation was identified and measured (Fig. 5B, left). Orthotopic xenograft tumor KLF6 downregulation is required for E2F1-mediated EMT and volumes of the shKLF6 group significantly increased as com- the metastatic phenotype paredwiththeshNCgroup(P < 0.001), whereas tumor To investigate the combined biologic effects of KLF6 and E2F1, volumes after E2F1 knockdown in the shNC group were greatly a Transwell assay was performed. As shown in Fig. 4A, the reduced (Fig. 5B, right). Histopathologic analysis of tumors suppressed KLF6 expression caused a robust increase in Caki-2 clearly revealed the interface between the tumor and normal cell migration and invasion. ShE2F1 introduction significantly kidney tissue (Fig. 5C). abrogated the invasive capacity of cancer cells transfected with The development of lung metastasis was evaluated after 786-O shKLF6. The E2F1 plasmid was ectopically transfected in OS-RC- cells were injected through the mouse tail vein in the different 2. Subsequently, KLF6 overexpression reversed the positive effect treatment groups. In this model, KLF6 knockdown significantly of E2F1 on cell migration and invasion, thereby suggesting that enhanced lung metastasis at 4 and 8 weeks after injection. shE2F1 KLF6 was involved in E2F1-mediated cell aggressiveness. The may counteract the metastasis-elevating effect of curtailed KLF6 abovementioned process was further examined by expression expression (Fig. 5D). All mice were sacrificed, and gross lung analysis of E-cadherin, N-cadherin, ZEB1, and vimentin, which specimens were collected for bioluminescence imaging (Fig. 5E). are potential vital genes that indicate the high malignancy of Furthermore, the presence of metastatic colonies in the lungs was cancer cells. N-cadherin, vimentin, and ZEB1 expression was validated by histologic analysis. The number of metastatic greatly upregulated after KLF6 knockdown, whereas impaired nodules in each group agreed with the bioluminescence intensity E-cadherin expression was found in Caki-2 cells. After the intro- detected in vivo (Fig. 5F). SN12-PM6 was introduced to establish

Figure 3. KLF6 binds to E2F1 promoter and directly represses the transcription of E2F1. A, Scheme revealed E2F1 as the top gene signature enriched in differentially expressed gene sets in primary metastatic phenotype. B, GSEA output drawing indicating the E2F1 enrichment in primary metastatic samples. C, Differences of E2F1 mRNA levels in primary metastatic and nonmetastatic ccRCC specimens. D, Close correlation of the mRNA levels of E2F1 and KLF6 in selected ccRCC samples. E, Basic mRNA expressions of ccRCC cell lines (left) and the inverse protein expressions of KLF6 and E2F1 in cell lines (right). F, Immunoﬂuorescent staining revealed the coexpressions of KLF6 and E2F1 in cellular nucleus. G, E2F1 mRNA levels were examined after KLF6 knockdown in 786-O or KLF6 overexpression in 769-P cell lines. I, ChIP assay was performed using anti-KLF6, normal rabbit IgG, and positive control. H, Putative KLF6-binding sites in E2F1 promoter and the nucleotide sequences in the right panel represent the predicted binding sequences, with the red capital letters indicating core binding elements. J, Luciferase assay was carried out to assess the effects of increasing amount of KLF6 on the luciferase activity of pGL3-Basic and pGL3- E2F1 plasmid in 293T cells. KLF6 overexpression levels were veriﬁed by Western blot analysis. K, Luciferase assay was performed to assess the effect of mutant (485/468) on the activity of pGL3-E2F1 reporter. , P < 0.05; , P < 0.01; , P < 0.001.

OF8 Cancer Res; 77(2) January 15, 2017 Cancer Research

KLF6 Transcriptionally Represses E2F1 in ccRCC Metastasis

Figure 4. E2F1 is critical for KLF6-mediated ccRCC migration and invasion. A, E2F1 knockdown signiﬁcantly reduced migration and invasion of Caki-2 cells transfected with shKLF6. On the contrary, KLF6 overexpression suppressed E2F1-mediated aggressiveness in OS-RC-2 cells. B, KLF6, E2F1, and EMT-related gene expressions were evaluated by Western blot assay in ccRCC cell lines. C, Morphologic alteration of cells transfected with the vectors as indicated. D, KLF6 knockdown in Caki-2 signiﬁcantly increased the amount of forming tubules. The angiogenic effect could be reversed by subsequent E2F1 abrogation. For OS-RC-2, inhibitory effect of KLF6 on microvessel forming could be reversed by E2F1 (P < 0.001). , P < 0.05; , P < 0.01; , P < 0.001. www.aacrjournals.org Cancer Res; 77(2) January 15, 2017 OF9

Gao et al.

Figure 5. Loss of KLF6 elevates metastatic potential of ccRCC cells in vivo by modulating E2F1. A, Representative bioluminescent pictures of nude mice that underwent orthotopic implantation with Luc-labeling 786-O cells stably transfected by indicated vectors (P < 0.001). B, Representative gross sample retrieved from the nude mice (left). The tumors were resected and measured (right). , P < 0.001. C, Hematoxylin and eosin staining of section from the orthotopic specimen. D, Representative bioluminescent images of nude mice injected via vein with Luc-labeling 786-O cells stably transfected by indicated vectors in 4 and 8 weeks, respectively (left). Measurement of bioluminescent signals of different groups (right). , P < 0.001. E, Representative gross view of the lungs with metastatic nodules of different groups (black arrows, metastatic nodules; left). The amount of the pulmonary metastatic lesions was counted in individual group (right). , P < 0.001. F, Hematoxylin and eosin stained slides of lungs dissected from nude mice from different groups. G, Representative bioluminescent images of nude mice bearing orthotopic implantation of Luc-labeling SN12-PM6 cells stably transfected by indicated plasmids in 3 months (left). Measurement of bioluminescent signals of different groups (right). , P < 0.001. H, IHC staining showed the changes in tubule formation in vivo following different vector transfection (, P < 0.01).

OF10 Cancer Res; 77(2) January 15, 2017 Cancer Research

KLF6 Transcriptionally Represses E2F1 in ccRCC Metastasis

Figure 6. Combined KLF6 and E2F1 expressions may predict the overall survival in patients with ccRCC. A, Inverse IHC staining of KLF6 and E2F1 in serial ccRCC sections. B, Correlation of KLF6 and E2F1 was statistically analyzed (P < 0.001). C, Close correlation of CD34 staining and KLF6 and E2F1 (P < 0.001). D, Overall survival of patients with ccRCC was calculated using Kaplan–Meier analysis according to low and high KLF6 staining. High KLF6 protein expression was determined by the strong KLF6 staining, whereas the rest were low KLF6 expression. The P values were calculated by using log-rank test.

the orthotopic and metastatic model, due to its pulmonary nostics GmbH (Supplementary Fig. S3). CD34 staining was metastatic property (22). ShE2F1 group mice exhibited a strong correlated closely with KLF6 and E2F1 staining in cancer tissues reversed effect (Fig. 5G). The in vitro assay proved that KLF6 and (Fig. 6C). The Kaplan–Meier analysis demonstrated that patients E2F1 affected tube formation, IHC staining was performed with with low KLF6 levels have poorer overall survival than those with the capillary-specific antibody CD34. Metastatic lesions of the high KLF6 levels (log-rank test; P ¼ 0.0079; Fig. 6D, left). For shKLF6 group presented more CD34 staining than the shNC samples grouped by the inverse expression of both proteins, group, whereas E2F1 depletion largely prevented tube formation patients with low KLF6 and high E2F1 had poorer overall survival (Fig. 5H). Therefore, the in vivo studies demonstrated that than those with high KLF6 and low E2F1 levels (log-rank test; P ¼ KLF6 repressed the metastatic properties of ccRCC cells by mod- 0.0003; Fig. 6D, right), with more significant differentiation. The ulating E2F1. coexpression and negative correlation of KLF6 and E2F1 in ccRCC tissues were observed and may predict overall survival. KLF6 expression is negatively correlated with E2F1 in ccRCC tissues and may predict overall survival The observed serial sections showed that KLF6 and E2F1 are Discussion mainly distributed in nuclei of the same cancer cells (Fig. 6A). We Cancer metastasis is a multistep process, wherein malignant analyzed the expression of both proteins by IHC with serial cells escape from the primary tumor to colonize distant sites (23). sections from the same tissue microarrays. Among the 114 ccRCC The overall mortality rates remain relatively constant because the specimens, strong KLF6 and E2F1 immunostaining was found in cancer was already at a late stage when the disease was diagnosed, 30.7% (35 of 114) and 65.8% (75 of 114) of the tumors, even with distant metastasis (24). However, the metastatic respectively. High levels of KLF6 expression were found in changes during disease progression are relatively unknown. 61.5% (24 of 39) of the tumors with low E2F1 staining, whereas Previous tissue profiling results suggested that KLF6 down- KLF6 presented low staining in 85.3% (64 of 75) of the tumors regulation and E2F1 activation are pivotal events in distant with high E2F1 expression. Statistical analysis with the Pearson c2 metastasis of ccRCC. Loss of KLF6 in a hepatocellular carcinoma test revealed the significant negative correlation between E2F1 mouse model results in increased carcinogenesis, promoted and KLF6 expression (P < 0.001, Fig. 6B). The quantification of metastasis to the lungs, and decreased survival (25). KLF6-SV1, protein expressions was confirmed in the platform of TissueG- an oncogenic splice variant of KLF6, was reported to increase

www.aacrjournals.org Cancer Res; 77(2) January 15, 2017 OF11

Gao et al.

metastatic potential in patients with lymph node–negative breast In summary, a novel genetic event was associated with ccRCC cancer. KLF6-SV1 overexpression in mammary epithelial cells metastasis. We identified KLF6 as a direct repressor of E2F1 and drove EMT and resulted in aggressive multiorgan metastasis demonstrated the novel function of KLF6–E2F1 in augmenting in vivo (26). The metastatic potential of KLF6-initiated metastasis EMT-related induction. The biologic significance of enhanced in ccRCC relied on a novel function of the classic transcription ccRCC progression and metastasis was highlighted by KLF6 factor E2F1. KLF6 directly modulated E2F1, which acted as an downregulation, which leads to the induction of E2F1 expression. EMT inducer. In addition, KLF6 depletion linked E2F1 activity to E2F1 can antagonize the tumor-suppressive function of KLF6 by the integrin EMT signaling pathway associated with cellular inhibiting cell proliferation, migration, invasion, and in vivo morphologic change and enhanced tumor cell malignancy. tumorigenesis. Sections of metastasized lung nodules were According to clinical data, a negative correlation was observed stained by CD34 to establish whether KLF6 depletion in the between the mRNA and protein expression of KLF6 and E2F1 in presence of high levels of E2F1 enhances the ability of the ccRCC ccRCC specimens. cell line to induce angiogenesis. As expected, the shKLF6-infected KLF6 expression is downregulated in various cancers, including 786-O cells significantly enhanced tubule formation, whereas non–small cell lung cancer (27), prostate cancer (7), and hepa- cells in the shKLF6 þ shE2F1 group prevented microvessel for- tocellular carcinomas (28). In addition, recent studies have asso- mation as compared with the shKLF6 group. This result suggested ciated the loss of KLF6 expression with poor clinical outcomes, that E2F1 prometastatic activity is strengthened when KLF6 cancer recurrence, and chemotherapeutic resistance (29–31). expression is abrogated. Unneglected, putative underlying mech- KLF6 has been previously suggested in OS-RC-2 cell line anism may occur considering that some tumors without EMT (ccRCC-derived) showing that stable expression of KLF6 lead to phenotype exhibited metastasis, further investigation is required reduced cell proliferation and cell-cycle arrest at G0–G1, correlat- to exclude the possibility that EMT may not be direct but an ing with an upregulation of p21 expression. It was the first study indirect factor associated with invasion or metastasis. showing the effect of KLF6 on proliferation of ccRCC-derived cells In total, we elucidated that the KLF6-mediated transcriptional (32). Ectopic KLF6 expression led to a G1 phase cell-cycle arrest, control of E2F1 expression maintains the invasive potential of thereby attenuating the cell proliferation. These findings cancer, which plays a pivotal role in the malignant progression of highlighted KLF6 as a tumor suppressor. human ccRCC and carries therapeutic implications. E2F1 prompts oncogenic and metastatic programs aside from its proliferative and apoptotic functions (33). E2F1 upregulation Disclosure of Potential Conflicts of Interest in mammary cell lines causes carcinogenesis and aggressive dis- No potential conflicts of interest were disclosed. tant metastasis in multiple cancers (34–36). E2F1 may enhance metastatic behavior in melanomas by directly binding with Authors' Contributions VEGFC and the corresponding receptor VEGFR-3 for transactiva- Conception and design: Y. Zhang, Q. Huang, X. Zhang tion (37). The oncogenic properties of E2F1 have been associated Development of methodology: Y. Zhang, Q. Huang, L. Wang, Y. Yao, X. Zhang with high EMT transition (38). However, the forces that drive Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Y. Gao, Y. Fan, D. Ni, X. Li, X. Zhang E2F1 gene expression in ccRCC remain unclear. in vitro Analysis and interpretation of data (e.g., statistical analysis, biostatistics, The study revealed that KLF6 mRNA and protein computational analysis): Y. Gao, X. Li, L. Gu, Y. Yao, Q. Ai, X. Zhang levels were upregulated, with a concomitant reduction of E2F1 Writing, review, and/or revision of the manuscript: Y. Gao, X. Ma, X. Li, Y. Yao, expressionuponKLF6transfection.KLF6upregulationabol- Q. Du, X. Zhang ished E2F1 expression, whereas KLF6 downregulation signifi- Administrative, technical, or material support (i.e., reporting or organizing cantly increased E2F1 expression. The previous document data, constructing databases): Y. Gao, X. Ma, D. Ni, Q. Huang, K. Liu, E. Song, X. Zhang demonstrated that E2F1 and KLF6 cooperated in activating the Study supervision: H. Li, X. Ma, Y. Zhang, X. Zhang DAPK2 promoter (39). In our study, promoter luciferase reporter and ChIP assays illustrated that KLF6 directly binds Grant Support to the human E2F1 gene promoter and inactivates it. In addi- This work was financially supported by National Nature Science Foundation tion, the siRNA-mediated repression of E2F1 in stable shKLF6- (No. 81572878 and 81402109), PR China and the National High Technology transfected cells reversed cell migration and metastatic pheno- Research and Development Program ("863"Program) of China: the screening types. E2F1 downregulation in vivo decreased the shKLF6-medi- and clinical validation of characteristic protein biomarkers in renal cancer based ated proliferation according to the orthotopic implantation on a large-scale biobank (2014AA020607) and clinical exploratory application model. We tested the role of KLF6 in ccRCC metastasis with of LESS and NOTES in urology (2012AA02101). The costs of publication of this article were defrayed in part by the payment of a nude mouse lung seeding assay. Compared with the control page charges. This article must therefore be hereby marked advertisement in shNC 786-O cells, the shKLF6-expressing cells developed more accordance with 18 U.S.C. Section 1734 solely to indicate this fact. lung metastases, which were completely diminished by E2F1 knockdown. Therefore, targeting E2F1 may be a promising Received February 17, 2016; revised October 7, 2016; accepted October 19, approach to avoid KLF6-mediated metastatic dissemination. 2016; published OnlineFirst October 25, 2016.

References 1. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer 3. Genetics of kidney cancer (renal cell cancer) (PDQ(R)): Health Profes- statistics, 2012. CA Cancer J Clin 2015;65:87–108. sional Version. In: PDQ cancer information summaries. Bethesda, MD: 2. Thomas JS, Kabbinavar F. Metastatic clear cell renal cell carcinoma: a review National Cancer Institute; 2002. of current therapies and novel immunotherapies. Crit Rev Oncol Hematol 4. Weinstock M, McDermott D. Targeting PD-1/PD-L1 in the treatment of 2015;96:527–33. metastatic renal cell carcinoma. Ther Adv Urol 2015;7:365–77.

OF12 Cancer Res; 77(2) January 15, 2017 Cancer Research

KLF6 Transcriptionally Represses E2F1 in ccRCC Metastasis

5. Randall JM, Millard F, Kurzrock R. Molecular aberrations, targeted therapy, 23. Pienta KJ, Robertson BA, Coffey DS, Taichman RS. The cancer diaspora: and renal cell carcinoma: current state-of-the-art. Cancer Metastasis Rev metastasis beyond the seed and soil hypothesis. Clin Cancer Res 2014;33:1109–24. 2013;19:5849–55. 6. Lievre A, Landi B, Cote JF, Veyrie N, Zucman-Rossi J, Berger A, et al. Absence 24. Dabestani S, Marconi L, Hofmann F, Stewart F, Lam TB, Canfield SE, et al. of mutation in the putative tumor-suppressor gene KLF6 in colorectal Local treatments for metastases of renal cell carcinoma: a systematic review. cancers. Oncogene 2005;24:7253–6. Lancet Oncol 2014;15:e549–61. 7. Liu X, Gomez-Pinillos A, Loder C, Carrillo-de Santa Pau E, Qiao R, Unger 25. Ahronian LG, Zhu LJ, Chen YW, Chu HC, Klimstra DS, Lewis BC. A novel PD, et al. KLF6 loss of function in human prostate cancer progression is KLF6-Rho GTPase axis regulates hepatocellular carcinoma cell migration implicated in resistance to androgen deprivation. Am J Pathol 2012;181: and dissemination. Oncogene 2016;35:4653–62. 1007–16. 26. Hatami R, Sieuwerts AM, Izadmehr S, Yao Z, Qiao RF, Papa L, et al. KLF6- 8. Kimmelman AC, Qiao RF, Narla G, Banno A, Lau N, Bos PD, et al. SV1 drives breast cancer metastasis and is associated with poor survival. Sci Suppression of glioblastoma tumorigenicity by the Kruppel-like transcrip- Transl Med 2013;5:169ra12. tion factor KLF6. Oncogene 2004;23:5077–83. 27. Ito G, Uchiyama M, Kondo M, Mori S, Usami N, Maeda O, et al. Kruppel- 9. Zhan L, Huang C, Meng XM, Song Y, Wu XQ, Miu CG, et al. Promising roles like factor 6 is frequently down-regulated and induces apoptosis in non- of mammalian E2Fs in hepatocellular carcinoma. Cell Signal 2014;26: small cell lung cancer cells. Cancer Res 2004;64:3838–43. 1075–81. 28. Vetter D, Cohen-Naftaly M, Villanueva A, Lee YA, Kocabayoglu P, Hanni- 10. Putzer BM, Engelmann D. E2F1 apoptosis counterattacked: evil strikes voort R, et al. Enhanced hepatocarcinogenesis in mouse models and back. Trends Mol Med 2013;19:89–98. human hepatocellular carcinoma by coordinate KLF6 depletion and 11. Muller H, Bracken AP, Vernell R, Moroni MC, Christians F, Grassilli increased messenger RNA splicing. Hepatology 2012;56:1361–70. E, et al. E2Fs regulate the expression of genes involved in differentiation, 29. Gehrau RC, D'Astolfo DS, Dumur CI, Bocco JL, Koritschoner NP. Nuclear development, proliferation, and apoptosis. Genes Dev 2001;15:267–85. expression of KLF6 tumor suppressor factor is highly associated with 12. Biswas AK, Johnson DG. Transcriptional and nontranscriptional functions overexpression of ERBB2 oncoprotein in ductal breast carcinomas. PLoS of E2F1 in response to DNA damage. Cancer Res 2012;72:13–7. One 2010;5:e8929. 13. Alla V, Engelmann D, Niemetz A, Pahnke J, Schmidt A, Kunz M, et al. E2F1 30. Teixeira MS, Camacho-Vanegas O, Fernandez Y, Narla G, DiFeo A, Lee B, in melanoma progression and metastasis. J Natl Cancer Inst 2010;102: et al. KLF6 allelic loss is associated with tumor recurrence and markedly 127–33. decreased survival in head and neck squamous cell carcinoma. Int J Cancer 14. Lee JS, Leem SH, Lee SY, Kim SC, Park ES, Kim SB, et al. Expression signature 2007;121:1976–83. of E2F1 and its associated genes predict superficial to invasive progression 31. Sangodkar J, Dhawan NS, Melville H, Singh VJ, Yuan E, Rana H, et al. of bladder tumors. J Clin Oncol 2010;28:2660–7. Targeting the FOXO1/KLF6 axis regulates EGFR signaling and treatment 15. Rosenfeldt MT, Bell LA, Long JS, O'Prey J, Nixon C, Roberts F, et al. E2F1 response. J Clin Invest 2012;122:2637–51. drives chemotherapeutic drug resistance via ABCG2. Oncogene 2014;33: 32. Cheng XF, Li D, Zhuang M, Chen ZY, Lu DX, Hattori T. Growth inhibitory 4164–72. effect of Kruppel-like factor 6 on human prostatic carcinoma and renal 16. Ma X, Gao Y, Fan Y, Ni D, Zhang Y, Chen W, et al. Overexpression of E2F1 carcinoma cell lines. Tohoku J Exp Med 2008;216:35–45. promotes tumor malignancy and correlates with TNM stages in clear cell 33. Engelmann D, Putzer BM. The dark side of E2F1: in transit beyond renal cell carcinoma. PLoS One 2013;8:e73436. apoptosis. Cancer Res 2012;72:571–5. 17. Zhu H, Wu K, Yan W, Hu L, Yuan J, Dong Y, et al. Epigenetic silencing of 34. Fang Z, Gong C, Liu H, Zhang X, Mei L, Song M, et al. E2F1 promote the DACH1 induces loss of transforming growth factor-beta1 antiprolifera- aggressiveness of human colorectal cancer by activating the ribonucleotide tive response in human hepatocellular carcinoma. Hepatology reductase small subunit M2. Biochem Biophys Res Commun 2015;464: 2013;58:2012–22. 407–15. 18. Zhao T, Ren H, Li J, Chen J, Zhang H, Xin W, et al. LASP1 is a HIF1alpha 35. Johnson JL, Pillai S, Pernazza D, Sebti SM, Lawrence NJ, Chellappan SP. target gene critical for metastasis of pancreatic cancer. Cancer Res 2015; Regulation of matrix metalloproteinase genes by E2F transcription factors: 75:111–9. Rb-Raf-1 interaction as a novel target for metastatic disease. Cancer Res 19. Huang QB, Ma X, Li HZ, Ai Q, Liu SW, Zhang Y, et al. Endothelial Delta-like 2012;72:516–26. 4 (DLL4) promotes renal cell carcinoma hematogenous metastasis. Onco- 36. Meier C, Spitschak A, Abshagen K, Gupta S, Mor JM, Wolkenhauer O, et al. target 2014;5:3066–75. Association of RHAMM with E2F1 promotes tumour cell extravasation by 20. Ma X, Fan Y, Gao Y, Zhang Y, Huang Q, Ai Q, et al. Dicer is down- transcriptional up-regulation of fibronectin. J Pathol 2014;234:351–64. regulated in clear cell renal cell carcinoma andin vitroDicer knockdown 37. Engelmann D, Mayoli-Nussle D, Mayrhofer C, Furst K, Alla V, Stoll A, et al. enhances malignant phenotype transformation. Urol Oncol 2014; E2F1 promotes angiogenesis through the VEGF-C/VEGFR-3 axis in a 32:46e9–17. feedback loop for cooperative induction of PDGF-B. J Mol Cell Biol 21. Ma X, Gu L, Li H, Gao Y, Li X, Shen D, et al. Hypoxia-induced over- 2013;5:391–403. expression of stanniocalcin-1 is associated with the metastasis of early stage 38. Knoll S, Furst K, Kowtharapu B, Schmitz U, Marquardt S, Wolkenhauer O, clear cell renal cell carcinoma. J Transl Med 2015;13:56. et al. E2F1 induces miR-224/452 expression to drive EMT through TXNIP 22. Ni D, Ma X, Li HZ, Gao Y, Li XT, Zhang Y, et al. Downregulation of FOXO3a downregulation. EMBO Rep 2014;15:1315–29. promotes tumor metastasis and is associated with metastasis-free survival 39. Britschgi A, Trinh E, Rizzi M, Jenal M, Ress A, Tobler A, et al. DAPK2 is a of patients with clear cell renal cell carcinoma. Clin Cancer Res 2014;20: novel E2F1/KLF6 target gene involved in their proapoptotic function. 1779–90. Oncogene 2008;27:5706–16.

www.aacrjournals.org Cancer Res; 77(2) January 15, 2017 OF13

KLF6 Suppresses Metastasis of Clear Cell Renal Cell Carcinoma via Transcriptional Repression of E2F1

Yu Gao, Hongzhao Li, Xin Ma, et al.

Cancer Res Published OnlineFirst October 25, 2016.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-16-0348

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2016/10/25/0008-5472.CAN-16-0348.DC1

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/early/2017/01/09/0008-5472.CAN-16-0348. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.