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Roles of KLF6 and KLF6-SV1in Ovarian Progression and Intraperitoneal Dissemination Analisa DiFeo,1Goutham Narla,1, 2 Jennifer Hirshfeld,1Olga Camacho-Vanegas,1Jyothsna Narla,6 Stephen L. Rose,7 Ta m a r a K a li r, 4 ShenYao,2 Alice Levine,2 Michael J. Birrer,8 Tomas Bonome,8 Scott L. Friedman,2 Richard E. Buller,7 and John A. Martignetti1, 3, 5

Abstract Purpose: We investigated the role of the KLF6tumor suppressor and its alternatively spliced isoform KLF6-SV1in epithelial (EOC). Experimental Design:We first analyzed tumors from 68 females with EOC for KLF6 gene inac- tivation using fluorescent loss of heterozygosity (LOH) analysis and direct DNA sequencing and then defined changes in KLF6and KLF6-SV1expression levels by quantitative real-time PCR.We then directly tested the effect of KLF6and KLF6-SV1inhibition in SKOV-3 stable cell lines on cel- lular invasion and proliferation in culture and tumor growth, i.p. dissemination, ascites production, and angiogenesis in vivo using BALB/c nu/nu mice. All statistical tests were two sided. Results: LOH was present in 59% of samples in a cell type ^ specific manner, highest in serous (72%) and endometrioid (75%) subtypes, but absent in clear cell tumors. LOH was significantly correlated with tumor stage and grade. In addition, KLF6expression was decreased in tumors when compared with ovarian surface epithelial cells. In contrast, KLF6-SV1 expression was increased f5-fold and was associated with increased tumor grade regardless of LOH status. Consistent with these findings, KLF6silencing increased cellular and tumor growth, angiogene- sis, and vascular endothelial expression, i.p. dissemination, and ascites production. Conversely, KLF6-SV1down-regulation decreased and invasion and completely suppressed in vivo tumor formation. Conclusion: Our results show that KLF6and KLF6-SV1are associated with key clinical features of EOC and suggest that their therapeutic targeting may alter ovarian cancer growth, progression, and dissemination.

Epithelial ovarian cancer (EOC) is the most lethal of all most women present late in disease course with widespread gynecologic and the fifth most frequent cause of cancer intra-abdominal metastasis. Thus, it will be critical to deaths in women (1). Although 5-year survival rates have characterize the genetic defects that underlie key steps in the increased over the past several decades to f40%, overall development and spread of ovarian cancer if advances are to be mortality rates remain relatively constant (1, 2) largely because made in diagnosis, prevention, and treatment. The tumor suppressor KLF6 is a member of the Kruppel-like family of involved in regulating differentiation, development, cellular proliferation, Authors’ Affiliations: Departments of 1Human Genetics, 2Medicine, 3Oncological growth-related , and (3, 4). KLF6 4 5 Sciences, Pathology, and Pediatrics, Mount Sinai School of Medicine, NewYork, inactivation has been implicated in several human cancers, New York; 6Department of Pathology, Regional Medical Center, San Jose, California; 7Holden Comprehensive Cancer, Division of Gynecologic Oncology,The including prostate (5, 6), colorectal (7), non–small cell lung University of Iowa Hospitals and Clinics, Iowa City, Iowa; and 8National Cancer (8), gastric (9), astrocytic glioma (10), nasopharyngeal (11), Institute, NIH, Bethesda, Maryland and hepatocellular (12, 13) carcinomas. Furthermore, de- Received 1/10/06; revised 4/10/06; accepted 4/19/06. creased KLF6 expression is associated with decreased patient Grant support: Department of Defense grant DAMD 17-03-1-0129 (J.A. Martignetti). survival in prostate (14, 15) and lung (16) cancers, whereas The costs of publication of this article were defrayed in part by the payment of page KLF6 overexpression decreases cell proliferation and reverts charges. This article must therefore be hereby marked advertisement in accordance tumorigenicity in glioblastoma cell lines (17). Depending on with 18 U.S.C. Section 1734 solely to indicate this fact. cell type and context, the growth-suppressive properties of KLF6 Note: Supplementary data for this article are available at Clinical Cancer Research have been associated with several highly relevant cancer Online (http://clincancerres.aacrjournals.org/). Current address for R.E. Buller: Oncology Medicine Development Centre, pathways, including -independent up-regulation of GlaxoSmithKline, Collegeville, PA. (5), disruption of cyclin D1 and cyclin-dependent kinase 4 Requests for reprints: John A. Martignetti, Department of Human Genetics, interaction (18), induction of apoptosis (8), and c-jun Mount Sinai School of Medicine, 1425 Madison Avenue, Box 1498, NewYork, NY inhibition (19). 10029. Phone: 212-659-6744; Fax: 212-849-2508; E-mail: John.Martignetti@ mssm.edu. Most recently, we have shown that a KLF6 single nucleotide F 2006American Association for Cancer Research. polymorphism associated with increased prostate cancer doi:10.1158/1078-0432.CCR-06-0054 risk increases alternative splicing of the gene into three

Clin Cancer Res 2006;12(12) June 15, 2006 3730 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2006 American Association for Cancer Research. KLF6andKLF6-SV1inOvarianCancer biologically active, cytoplasmic isoforms, KLF6-SV1, KLF6-SV2, (30 seconds), 55jC (30 seconds), and 72jC (1 minute) for 45 cycles, and KLF6-SV3, in both normal and cancerous tissues (20). and a final extension of 72jC (5 minutes). Splice variant expression is up-regulated in prostate cancer Real-time PCR analysis. For quantitating target , tumors compared with normal prostate tissue (20) and the RNA isolation from cultured cells and tumor xenografts was done using RNeasy Mini and Midi kits (Qiagen). All RNA was treated with activity of one of the splice isoforms KLF6-SV1 directly opposes DNase (Qiagen). RNA (1 Ag) was reverse transcribed for each reaction KLF6 effects on cell proliferation, colony formation, invasion, using first-strand cDNA synthesis with random primers (Promega, in vivo and tumor growth (21). Madison, WI). mRNA levels were quantified by quantitative real-time In view of these combined findings, we investigated the role PCR (qRT-PCR) using the following PCR primers on an ABI PRISM of KLF6 and KLF6-SV1 in ovarian cancer. Most EOC tumor 7900HT (Applied Biosystems): KLF6 forward 5¶-CGGACGCACACAG- samples have KLF6 allelic loss, decreased wild-type expression, GAGAAAA-3¶ and reverse 5¶-CGGTGTGCTTTCGGAAGTG-3¶, total KLF6 and increased expression of the dominant-negative KLF6-SV1 forward 5¶-CTGCCGTCTCTGGAGGAGT-3¶ and reverse 5¶-TCCACA- isoform. Targeted reduction of KLF6 in the ovarian cancer cell GATCTTCCTGGCTGTC-3¶, glyceraldehyde-3-phosphate dehydrogenase ¶ ¶ ¶ line SKOV-3 resulted in marked increases in cell proliferation, forward 5 -CAATGACCCCTTCATTGACC-3 and reverse 5 -GATCT- ¶ ¶ invasion, tumor growth, angiogenesis, and i.p. dissemination CGCTCCTGGAAGATG-3 , KLF6-SV1 forward 5 -CCTCGCCAGGGAAG- GAGAA-3¶ and reverse 5¶-TCCACAGATCTTCCTGGCTGTC-3¶, KLF6-SV2 in-vivo, modeling late stage EOC. In addition, these changes forward 5¶-TCGGGGAAGCCAGGAGAA-3¶ and reverse 5¶-TCCACA- were associated with marked increases in vascular endothelial GATCTTCCTGGCTGTC-3¶, KLF6-SV3 forward 5¶-CGGACGCACACA- in vivo growth factor (VEGF) both in culture and . In contrast, GGTGTT-3¶ and reverse 5¶-TCCACAGATCTTCCTGGCTGTC-3¶, VEGF KLF6-SV1 reduction resulted in decreased proliferation, forward 5¶-CCGCAGACGTGTAAATGTTCCT-3¶ and reverse 5¶-CGG- invasion, angiogenesis, and tumorigenicity. Collectively, our CTTGTCACATCTGCAAGTA-3¶, and c- forward 5¶-CAGCTGCTTA- results reveal an important association between KLF6 and GACGCTGGATTT and reverse 5¶-ACCGAGTCGTAGTCGAGGTCAT. KLF6-SV1 and the molecular basis of ovarian carcinogenesis. Primer sequences for FLT-1, KDR, platelet/endothelial cell adhesion molecule, TIE1, VE-cadherin, and ANG2 have been described previously (23). All values were calculated by normalizing the levels of each target for Materials and Methods each cDNA to glyceraldehyde-3-phosphate dehydrogenase and then using this normalized value to calculate fold change compared with Tumor samples, preparation, and DNA isolation. Tumor specimens control. KLF6 splicing fold change was calculated by dividing the fold were collected and analyzed under institutional review board approval. changes in total KLF6 (KLF6 + alternatively spliced KLF6 transcripts) by Histologic diagnosis was validated by pathology review at the that in KLF6 alone. All experiments were done in triplicate and repeated University of Iowa Institutional Gynecological Oncology Tumor Board. three independent times. The expression level of each amplicon was Tumors were staged in accordance with the International Federation of calculated by normalizing each cDNA to glyceraldehyde-3-phosphate Gynecology and Obstetrics surgical staging system. Tumor samples were dehydrogenase and then using this normalized value to calculate fold snap frozen at the time of surgery in liquid nitrogen. DNA isolation and change. All experiments were done at least thrice and each in triplicate. preparation techniques have been reported previously (22). Ten Statistical significance was determined by one-way ANOVA using a additional samples were collected and analyzed from the Regional Bonferroni correction. Medical Center (San Jose, CA). Normal DNA was obtained from Ovarian surface epithelium gene expression analysis. A set of 10 microdissected tissue in the region of the tumor or from margins with normal ovarian surface epithelium cytobrushing specimens was no evidence of cancer. In all cases, a 5-Am H&E section was used as a obtained from the normal ovaries of donors during surgery for other histologic reference for normal and tumor-derived tissues. Manual gynecologic diseases at Brigham and Women’s Hospital according to an microdissection was done on serial 20-Am sections, and DNA was institutional review board–approved protocol as described previously subsequently extracted using commercial reagents (Ambion, Austin, (24). Total RNA was subsequently isolated using a RNeasy Micro TX). Normal whole ovarian tissue cDNA was obtained from a (Qiagen). All purified total RNA specimens were quantified and commercial resource (Clontech, Mountain View, CA). checked for quality with a Bioanalyzer 2100 System (Agilent, Palo Loss of heterozygosity and DNA analysis. Fluorescent loss Alto, CA) before further manipulation. of heterozygosity (LOH) analysis using genomic DNA from matched As described previously, two rounds of amplification were used to normal/tumor ovarian tissue and markers has been described previ- generate sufficient product from 25 ng ovarian surface epithelial total ously (5). The exponential range of the PCR varied between 30 and 38 RNA for Affymetrix (Santa Clara, CA) GeneChip analysis (25). Briefly, cycles and was specifically determined for each marker and sample. total RNA was reverse transcribed using the Two-Cycle cDNA Data were analyzed using ABI Genescan and Genotyper software Synthesis kit (Affymetrix) and oligo-dT24-T7 (5¶-GGCCAGTGAATTG- packages (Perkin-Elmer, Boston, MA) and allelic loss was scored by two TAATACGACTCACTATAGGGAGGCGG-3¶) primer according to the independent observers. A relative allele ratio of <0.7, correlating with an manufacturer’s instructions followed by amplification with the allele loss of f40%, was defined as LOH (5). Samples that had either MEGAscript T7 kit (Ambion). After cleanup of the cRNA, second loss of at least one of the KLF6-specific markers flanking the KLF6 gene, round double-stranded cDNA was generated and amplified using the M1, M2, and M4, or a flanking marker when the contiguous KLF6 IVT labeling kit (Affymetrix). cRNA was hybridized to a U133 Plus 2.0 specific marker was noninformative were regarded as having LOH. All oligonucleotide array (Affymetrix) and stained with phycoerythrin- sample marker combinations were analyzed at least twice. conjugated streptavidin (Molecular Probes, Eugene, OR) in a Fluidics PCR products were directly sequenced, following purification Station 450 (Affymetrix). The arrays were then scanned using a confocal (QIAquick PCR purification kit, Qiagen, Valencia, CA), on an ABI laser GeneChip Scanner 3000 with GeneChip Operating Software PRISM 3700 automated DNA analyzer (Applied Biosystems, Foster City, (Affymetrix) and normalized to a target value of 500 using MAS 5.0. CA) and data were analyzed using the program Sequencher (Gene Codes Normalized data were uploaded into the National Cancer Institute Corp., Ann Arbor, MI). The following sets of intronic primers (sense and Microarray Analysis Database for quality-control screening and collation antisense, respectively) were used to amplify the coding region and of KLF6 and housekeeping gene (GUSB, ACTA2, ACTB, PPIH, and PPIA) intron/exon boundaries of KLF6 exon 2: forward 5¶-CGGGCAGCAAT- signal intensities. The expression values for KLF6 and the housekeeping GTTATCTGTCCTTC-3¶ and reverse 5¶-CCCTCCAGGGCTGGTGCA-3¶. were called present for all of the arrays according to the MAS PCR cycling conditions were 94jC (10 minutes) for 1 cycle, 94jC 5.0 algorithm.

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Cell culture and transfection. SKOV-3 cells were purchased from Results the American Type Culture Collection (Manassas, VA). Stable cell lines were generated by cotransfection of the pSUPER-siRNA- KLF6 LOH and decreased expression in EOC. We explored luciferase (si-Luc), pSUPER-si-KLF6, and pSUPER-si-SV1 with a the role of KLF6 in EOC by first determining the frequency of puromycin expression plasmid and selected with 2 Ag/mL puromycin as described previously (21). Polyclonal pools of each small LOH at the KLF6 locus on 10p15 and interrogat- interfering RNA (siRNA) –infected cell lines were collected and ing tumor-derived DNA for possible in a set of KLF6 and KLF6-SV1 knockdown was determined by qRT-PCR and clinical samples. The clinicopathologic profiles of these samples Western blot. (Table 1) are representative of the varied clinical spectrum of Western blot analysis and densitometricanalysis. Cell extracts were EOC wherein International Federation of Gynecology and harvested in radioimmunoprecipitation assay buffer (Santa Cruz Obstetrics stage III, grade 3, serous ovarian carcinomas Biotechnology, Santa Cruz, CA; standard protocol). Tumor tissue dominate as the most frequent presentation of advanced EOC. extracts were harvested and prepared in the T-PER reagent (Pierce, A total of 68 paired EOC and normal samples were assayed for A Rockford, IL). Equal amounts of (50 g; determined by LOH using six microsatellite markers: KLF6M1, KLF6M2, and the Bio-Rad DC protein quantification assay, Hercules, CA) were KLF6M4, which tightly flank the KLF6 gene locus (5), and loaded and separated by PAGE and transferred to nitrocellulose D10S249, D10S594, and D10S591, which are more distal. This membranes. Actin (SC-1616), KLF6 (SC-7158), and VEGF (SC-507) marker set rendered all tumor samples informative for at least were obtained from Santa Cruz Biotechnology, c-myc (OP10) was from (Carpinteria, CA) and one marker at the KLF6 locus (Fig. 1A). Overall, 59% (40 of 68) proliferating cell nuclear antigen (M-0879) antibody from DAKO of the EOC samples showed LOH of the KLF6 locus. In addition, (Carpinteria, CA). LOH was subtype specific, being present in 72% of serous Enhanced chemiluminescent immunoblot images were analyzed by carcinomas (33 of 46), 75% of endometrioid tumors (6 of 8), scanning densitometry and quantified with a BIOQUANT NOVA and 11% of mucinous EOCs (1 of 9). None of the 5 clear cell imaging system. Values were expressed as fold change relative to carcinomas showed LOH (Table 2). Moreover, KLF6 LOH was control and normalized to actin as a loading control. significantly correlated with tumor International Federation of Analysis of proliferation. Proliferation was determined by measur- P P 3 Gynecology and Obstetrics stage ( < 0.05) and grade ( < 0.05; ing [ H]thymidine incorporation. The siRNA SKOV-3 stable cell lines Supplementary Fig. S1). containing si-Luc, si-KLF6, or si-SV1 were plated at a density of KLF6 mutation status was examined by direct sequencing of 50,000 cells per well in 12-well dishes. Forty-eight hours after plating, 1 ACi/mL [3H]thymidine (Amersham, Piscataway, NJ) was exon 2 in all samples. This exon encodes three quarters of the added. After 2 hours, cells were washed four times with ice-cold PBS wild-type protein and contains the majority of mutations and fixed in methanol for 30 minutes at 4jC. After methanol identified previously in other human cancers (5–7, 9, 10, 12, removal and cell drying, cells were solubilized in 0.25% NaOH/ 13). In contrast to previous findings in prostate (5, 6), colorectal 0.25% SDS, samples were then neutralized with hydrochloric acid (7), hepatocellular carcinomas (12), gastric (9), glioma (10), (1 N), and disintegrations per minute were measured by liquid scintillation counting (5). Invasion assays. Standard invasion assays were done in Boyden chambers using a reconstituted basement membrane (Matrigel, 0.5 mg/mL). Coated membranes were first blocked with 0.5% bovine Ta bl e 1. Clinical profile of patient tumor samples, serum albumin/DMEM and equilibrated in 0.1% bovine serum n (%) albumin/DMEM. Approximately 105 cells in serum-free DMEM were added to the upper chamber and conditioned medium was derived Histologic type from NIH 3T3 fibroblasts in the lower chamber as a chemoattractant. Following incubation for 19 hours at 37jC, cells in the upper Epithelial chamber were thoroughly removed by suctioning. Cells that had Serous carcinoma 46(68) invaded through the barrier were fixed in 10% formalin and stained Endometrioid 8 (12) with 4¶,6-diamidino-2-phenylindole in PBS. Nuclei were visualized Mucinous carcinoma 9 (13) under a fluorescence microscope and images of five randomly selected Clear cell 5 (7) nonoverlapping fields were counted (16). Three independent experi- Histologic grade ments were done and statistical significance was determined by one- 15(7) way ANOVA with a Bonferroni correction. 219(28) Tumorigenicity and i.p. dissemination assays. Stable siRNA SKOV-3 cells (1  107) were injected into the left flank of female 6- to 8-week- 344(65) old BALB/c nu/nu mice. Tumor volume was assessed weekly and FIGO stage determined using the formula: length  width  width  0.4. After 6 I7(10) weeks, mice were sacrificed and tumors were removed for RNA and II 4 (6) protein analysis. For i.p. dissemination experiments, female 6- to III 39 (57)  7 8-week-old BALB/c nu/nu mice were injected i.p. with 1 10 stable IV 18 (26) siRNA SKOV-3 cells and the abdominal circumferences were measured Primary residual tumor weekly until week 6 when the mice were sacrificed and tumors and None 15 (22) ascitic fluid were collected. All animal studies were approved by the V2cm 20(29) Mount Sinai School of Medicine IACUC. 2cm 19(28) VEGF ELISA. VEGF levels from both conditioned medium and > tumor xenograft samples were quantified using a commercially avail- No data 14 (2 1) able ELISA kit using the manufacturer’s recommended protocol (Quantikine Human VEGF Immunoassay kit, R&D Systems, Minne- Abbreviation: FIGO, International Federation of Gyncology and Obstetrics. apolis, MN).

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and nasopharyngeal (11) carcinomas, no somatic mutations compared with normal ovarian tissue (P < 0.01; Fig. 1B; were detected in any of the EOC samples analyzed. average levels of wild-type KLF6 are 0.2 F 0.11 in LOH We next explored whether allelic loss was associated with samples, 1.2 F 0.2 in no LOH samples, and 1.5 F 0.23 in decreased KLF6 mRNA expression. Fifteen of the 26 tumor normal ovarian tissues). KLF6 expression levels in tumors samples showing LOH that had RNA available for analysis without LOH trended lower but were not significantly had, on average, an 80% reduction in expression when different when compared with a panel of cDNAs isolated from compared with tumors without LOH (P < 0.001) or when normal whole ovaries (Fig. 1B). These findings showing

Fig. 1. A, LOH status of KLF6. LOHof the KLF6 locus was analyzed using microsatellite markers (Yaxis) from the10p15 region and KLF6-specific markers KLF6M1,KLF6M2, and KLF6M4, which have been described previously (5). Cases (Xaxis) are ranked according to degree of loss with respect to KLF6and separated by histologic type. Black squares, LOH; gray squares, noninformative; white squares, no evidence of loss; -, a PCR that failed three or more times. Tumors were defined as having LOH if one or all of the markers flanking the KLF6locus were lost. B, correlation of LOH and RNA levels. KLF6mRNA levels in 26samples were analyzed by qRT-PCR and correlated to their respective LOH status. All samples with LOH (n = 15) showed f80% down-regulation in KLF6mRNA levels when compared with tumors ( n = 9) without LOH (P < 0.001) or when compared with normal whole ovary tissue 9 (n =5;P < 0.01). C, KLF6expression in ovarian surface epithelium ( OSE) brushings compared with select housekeeping genes. KLF6microarray signal intensity values were averaged for 10 ovarian surface epithelium brushings and compared with average ex pression levels of 5 housekeeping genes: GUSB, ACTA2, ACTB, PPIH, and PPIA . KLF6expression was comparable with housekeeping gene levels, indicating that KLF6is reliably expressed in ovarian epithelial cells. D, KLF6expression was decreased in 62(82%) tumor samples when compared with normal ovarian surface epithelial cells.The overall decrease was 1.66-fold. Statistical analysis was done using a Student’s t test with two-tailed distribution and two-sample equal variance. *, P < 0.05; **P < 0.01; ***P < 0.001.

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amount of splice variants in each tumor sample, we first Ta bl e 2 . Summary of allelic loss of KLF6in EOC determined the ‘‘KLF6 splicing index’’: the ratio of all KLF6 transcripts compared with only wtKLF6. We used qRT-PCR Histologic type Allelic loss of 10p15 (%) primers specific to either conserved or unique regions within the Serous 33/46(72)* KLF6 transcript such that only KLF6 or both wild-type and Endometrioid 6/8 (75)* variant transcripts (total) were amplified (20). We detected a Mucinous 1/9 (11) 2-fold increase in KLF6 splicing index in poorly differentiated Clear cell 0/5 (0) grade III tumors compared with well to moderately differenti- To t a l 4 0 / 68 (5 9 ) ated grade I or II tumors (P < 0.05; Fig. 2B; average KLF6 splicing index is 1.2 F 0.06 in grade I/II tumors versus 1.8 F 0.11 in grade *Allelic loss of KLF6was significantly more frequently found in serous and III tumors). Additionally, a 30% increase in KLF6 alternative endometrioid carcinoma (P < 0.05). splicing was detected in stage III/IV tumors when compared with stage I/II, but due to the low number of stage I/II tumors statistical significance was not achieved. Differences in splicing decreased expression levels with allelic loss are in accord were also evident when comparing serous with nonserous with previous studies in non–small cell lung cancer wherein tumors. Serous tumors had a 50% increase in the splicing index 14of 14samples with LOH had lower KLF6 expression (P < 0.05; data not shown). levels (8). Targeted down-regulation of KLF6 and KLF6-SV1 alters Finally, given that the vast majority of ovarian cancer is cellular proliferation and invasion. Having identified correla- believed to arise from ovarian surface epithelial cells, we also tions between KLF6 family members and clinicopathologic compared the level of KLF6 expression in these cells to EOC. variables in patient samples, we next directly explored the KLF6 expression was decreased in most high-grade ovarian biological role of KLF6 and KLF6-SV1 using siRNA-mediated cancers. Specifically, KLF6 expression in a set of 10 ovarian gene silencing to specifically down-regulate their expression. surface epithelial cells was compared with its expression We used the human serous cystadenocarcinoma-derived SKOV- across 76 stage III EOC samples by Affymetrix GeneChip 3 cell line, which is capable of inducing i.p. carcinomatosis in analysis (24, 25). As shown in Fig. 1C and D, KLF6 xenograft models and generated stable cell lines expressing expression was decreased in 62 (82%) tumor samples when siRNAs to luciferase (si-Luc), wild-type KLF6 (si-KLF6), and compared with ovarian surface epithelial cells and the overall KLF6-SV1 (si-SV1). In their respective cell lines, KLF6 protein decrease was 1.66-fold (P < 0.05). Taken together, these findings showing KLF6 LOH and/or decreased expression in most samples are consistent with its role as a tumor suppressor in EOC. KLF6 gene splicing is up-regulated in ovarian tumors. Based on our previous identification of increased alternative splicing of the KLF6 gene into biologically active KLF6 alternative splice forms in prostate tumors (20), we next explored whether these variants were also expressed in ovarian tissue and whether KLF6 alternative splicing was dysregulated in EOC. RNA from 33 tumors and a panel of 5 normal ovarian samples were analyzed by qRT-PCR using highly specific and sensitive sets of PCR primers for each KLF6 isoform (21). All but one of the cancer samples, 32 of 33 (97%), expressed KLF6-SV1 and 25 of 33 (75%) expressed KLF6-SV2 alternative splice forms. KLF6-SV3 was not detected in any of the samples analyzed. Most importantly, KLF6-SV1 expression was increased, on average, 5-fold (P < 0.001) in EOC samples with LOH relative to normal ovarian tissue and f3-fold in those tumors without LOH (SV1, 0.08 F 0.04in normal tissue, 0.22 F 0.03 in no LOH samples, and 0.58 F 0.04in LOH samples). Increased KLF6-SV1 expression was indepen- dent of LOH status (P < 0.01; Fig. 2A) and was particularly notable in view of the overall decrease in KLF6 expression in EOC (Fig. 1B). KLF6-SV1 overexpression correlates with advanced tumor Fig. 2. A, increased KLF6-SV1expression in ovarian tumors.Tumors with LOH grade. We next explored possible correlations between in- had a 5-fold increase in KLF6-SV1compared with normal tissue (P < 0.001) and creased KLF6 alternative splicing and EOC clinicopathologic tumors without LOH had a 2-fold up-regulation (P < 0.01). KLF6-SV2 expression remained relatively constant in all tumors. B, KLF6splicing ratio correlates with factors, including tumor grade, histology, and International tumor grade. qRT-PCR was done on a total of 30 ovarian cancer samples of various Federation of Gynecology and Obstetrics stage. Our findings grades (11grade I/II and 19 grade III).The ratio of total to wild-type KLF6was suggest that increased KLF6 isoform expression is associated then determined as in (A). High-grade tumors were associated with an f2-fold increase in splicing ratios when compared with grade I/II tumors (P < 0.05). with more aggressive tumors and may be specific to the Statistical analysis was done using a Student’s t test with two-tailed distribution histologic tumor type. To quantitatively assess the relative and two-sample equal variance. *, P < 0.05; **P < 0.01; ***P < 0.001.

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Fig. 3. Effect of siRNA-mediated decreases in wild-type and KLF6-SV1on cell proliferation and invasion. The degree of wild-type and KLF6-SV1silencing was first determined on the RNA and protein levels in SKOV-3 cell lines as shown in (A and B), respectively. C, cellular proliferation in siRNA stable cells. si-KLF6 SKOV-3 cells showed an f50% increase in cell proliferation when compared with control cells (P < 0.001), whereas si-KLF6-SV1cells showed an f40% decrease in proliferation (P <0.01). D, invasion capacity in siRNA stable cells. To p, representative photomicrographs of the underside of a Matrigel insert stained with 4¶,6-diamidino-2-phenylindole; bottom, number of cells per Â400 magni- fied visual field. Reduction of KLF6-SV1 resulted in a 60% decrease in cell invasion compared with control (P < 0.01), whereas cells with decreased KLF6levels showed a 2.5-fold increase in cellular invasion (P < 0.001). Cell number was determined from analysis of four representative visual fields from three independent experiments. Statistical analysis was done using a Student’s t test with two-tailed distribution and two-sample equal variance. *, P < 0.05; **P < 0.01; ***P < 0.001.

levels were reduced f50%, whereas KLF6-SV1 was decreased antigen, were consistently up-regulated (P < 0.01; Fig. 4C). 75% compared with si-Luc control (Fig. 3A and B). No further experiments could be done on si-SV1-expressing Our findings show that KLF6 and KLF6-SV1 affect cellular tumors because they completely regressed. proliferation and invasion of the SKOV-3 cell line. We first Targeted KLF6 and KLF6-SV1 reduction alters VEGF expression examined cellular proliferation differences between these siRNA and secretion. Given the striking effects of targeted KLF6 and stable cell lines. In the si-KLF6-expressing cell line, proliferation KLF6-SV1 reduction on in vivo tumor growth, we examined was increased f2-fold (P < 0.001). Conversely, targeted reduc- their effect of reducing KLF6 and KLF6-SV1 on VEGF tion of KLF6-SV1 resulted in a 25% reduction in proliferation expression and secretion, because VEGF is a critical regulator (P < 0.01; Fig. 3C). Unexpectedly, given our previous findings of angiogenesis and EOC tumor development (26). Differ- in si-KLF6 and si-SV1 prostate cancer cell lines (5, 20) but ences in all four transcripts encoding monomeric VEGF, similar to findings in non–small cell lung cancer (8), decreases excluding isoform 121, were examined by qRT-PCR using in KLF6 and KLF6-SV1 had no effect on either p21 RNA or primers spanning exons 7 and 8 of the VEGF gene. Reduction protein levels, suggesting a tissue- or cancer-specific effect (data of KLF6 expression led to increased VEGF expression and not shown). secretion (P < 0.01; Fig. 5A). In contrast, KLF6-SV1 down- We next tested the effects of KLF6 and KLF6-SV1 silencing on regulation decreased secreted VEGF by 60% to 80% (P < 0.01; invasive capacity. Targeted KLF6 reduction resulted in a 2.5-fold Fig. 5A). increase (P < 0.001) in cellular invasion, whereas KLF6-SV1 We next determined whether these effects in cultured cells down-regulation had the opposite effect, decreasing invasion by were also sustained in vivo and were associated with tumor 60% (P < 0.001; Fig. 3D). growth differences in the mouse xenograft model. Consistent KLF6 and KLF6-SV1 directly affect tumor mass and growth with our in vitro studies, there was a significant increase in rate in vivo. SKOV-3 stable cell lines expressing si-Luc, si- both VEGF mRNA (P < 0.001) and protein levels in the si- KLF6, or si-SV1 were s.c. injected into nude mice, monitored KLF6-derived tumors (Fig. 5B and C). On average, si-KLF6- weekly for tumor growth, and then sacrificed at the end of 6 derived tumors expressed f15-fold higher VEGF concentra- weeks. Most conspicuously, all mice transplanted with si-SV1 tion (P < 0.01; Fig. 5C). In further support of these findings, a cells failed to form persistent tumors. The small tumors that panel of angiogenesis-related genes, consisting of ANG1, initially developed regressed after 3 weeks (Fig. 4A). In ANG2, FLT-1, KDR, TIE1, VE-cadherin, and platelet/endothe- contrast, reduction of KLF6 doubled tumor growth rate and lial cell adhesion molecule-1, shown previously to more mass, consistent with its tumor-suppressive functions (P < accurately reflect angiogenesis than single marker genes alone 0.01; Fig. 4A and B). In these tumors, two known markers of (23), were all significantly increased in si-KLF6-derived tumors cellular proliferation, c-myc and proliferating cell nuclear (Fig. 5D).

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Decreased KLF6 increases i.p. dissemination in vivo. Finally, levels were comparable with those reported in EOC ascites the role of KLF6 on EOC progression was examined in an fluid from patients (29). In marked contrast, no overt in vivo ovarian cancer mouse model that recapitulates metastatic tumors or ascitic fluid collections were observed advanced-stage disease with i.p. carcinomatosis and ascites in the si-Luc control mice at this time point. production (27, 28). Combined observations on patient samples and experimental studies all suggested that KLF6 loss might contribute to metastasis because it was associated with Discussion increasing EOC stage and grade. The SKOV-3 si-Luc or si- KLF6 cell lines were injected into the peritoneal cavity of Our findings in patient samples and experimental models BALB/c nu/nu mice and tumor growth was followed by define a high frequency of KLF6 allelic loss, decreased KLF6 weekly measurement of abdominal circumferences. All si- expression, and increased expression of the dominant-negative KLF6 mice developed pronounced abdominal swelling from isoform KLF6-SV1 and suggest that dysregulation of KLF6 and ascites accumulation and i.p. carcinomatosis at 6 weeks post- KLF6-SV1 may be an important event in the development and tumor cell inoculation, at which point animals from both spread of EOC. Mounting evidence in other tumors has groups were euthanized and tumor burdens were assessed. In highlighted a variety of KLF6-inactivating mechanisms relevant the si-KLF6 mice, tumors were present on the peritoneal, to tumor growth and spread. These mechanisms include (a) intestinal, and diaphragmatic surfaces (Fig. 6A). Average LOH and somatic mutation in prostate (5, 6), colorectal (7), ascites volume was 600 AL (Table 3), with an average VEGF malignant glioma (10), nasopharyngeal carcinomas (11), concentration of 870 pg/mL (data not shown). These VEGF HCC (12, 13), and gastric cancer (9); (b) transcriptional

Fig. 4. Divergent effects of stably expressed siRNAs to KLF6and KLF6-SV1on tumor growth in vivo. Stable SKOV-3 cell lines expressing siRNAs to either luciferase (control; n =9),wild-typeKLF6(n =15),orKLF6-SV1(n = 5) were injected into nude mice and their growth was assessed over a 6-week . A and B, reduction of KLF6 (red line) resulted in an approximate doubling of tumor growth rate and burden (P < 0.01). Strikingly, reduction of KLF6-SV1 (green line) resulted in decreased tumor growth rates and tumor regression after 3 weeks. C, changes in tumor growth are reflected in c-myc and proliferating cell nuclear antigen levels. qRT-PCR (left)ofc-mycRNA and Western blot analysis of c-myc and proliferating cell nuclear antigen (PCNA)proteins(right) isolated mouse xenograft tumors was done. Increased tumor growth was paralleled by an increase in these proliferation markers (P < 0.05). Statistical analysis was done using a Student’s t test with two-tailed distribution and two-sample equal variance. *, P < 0.05; **P < 0.01; ***P < 0.001.

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Fig. 5. Divergent effects of stably expressed siRNAs to KLF6and KLF6-SV1onVEGF secretion in cultured cells and in vivo. A, VEGF mRNA and protein levels were determined in stable SKOV-3 cell lines grown in culture, expressing siRNAs to either luciferase, wild-type KLF6, or KLF6-SV1.Reduction of KLF6 and KLF6-SV1resulted in increases and decreases, respectively,ofVEGFmessage (P < 0.01and P < 0.01; top left) and the189 and165secreted amino acidisoforms (top right).VEGFconcentrations were also confirmed in conditioned medium by ELISA (bottom). B and C, analysis ofVEGF mRNA and protein isoforms in tumor xenografts. Decreased KLF6expression is associated with i ncreases in bothVEGFmessage (P <0.001; B, top ) and protein levels as shown by bothWestern blot (B, bottom)andELISA(C). D, up-regulation of angiogenesis-related genes in si-KLF6-derived tumors. qRT-PCRof six markers of angiogenesis shows that decreased levels of KLF6 expression are associated with statistically significant increases in their expression. Statistical analysis was done using a Student’s t test with two-tailed distribution and two-sample equal variance. *, P < 0.05; **P < 0.01;***P < 0.001.

silencing through hypermethylation in esophageal expression profiles (32–34) between clear cell and the other cancer cell lines (30); and (c) dysregulated alternative splicing histologic subtypes. in prostate cancer (20). This study is the first to identify loss Our findings also show the importance and prevalence of and dysregulation of KLF6 and KLF6-SV1 in ovarian cancer KLF6-SV1 up-regulation in EOC tumor proliferation. We development. Of particular interest, therefore, is a recent microarray study that associated decreased KLF6 expression with cisplatin resistance in ovarian cancer cell lines (31). Taken together, these results suggest that not only is KLF6 loss a frequent event but also this loss may also be predictive of staging and long-term outcome. Indeed, reports in prostate and lung cancers have identified decreased KLF6 expression as a predictor of survival (14–16). Future studies are therefore warranted to examine this question. In our study, two of the most common EOC histologic subtypes, serous papillary adenocarcinoma and endometrioid carcinoma, accounting for >70% of all EOC tumors, had the Fig. 6. I.p. model of tumor growth and dissemination. Stable SKOV-3 cell lines highest LOH frequency, 72% and 75%, respectively. Interest- expressing siRNAs to either luciferase (control; n = 9) or wild-type KLF6( n =15) ingly, KLF6 was not lost in clear cell samples. This genetic were injected into the peritoneal cavity of nude mice and their growth was assessed over an 6-week period. Metastatic tumors to the peritoneal, intestinal, and dia- variance between subtypes is consistent with other reports phragmatic surfaces were present in all si-KLF6mice. There was no overt evidence showing differences in p53 mutation/LOH status (32) and gene of dissemination in control si-Luc mice.

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significant that in vivo s.c. tumors expressing si-KLF6 were 2- Table 3. Summary of in vivo tumor growth and fold larger, had extensive ascites, expressed 5-fold more VEGF, ascites accumulation in ovarian cancer mouse models and, on average, had an 8-fold increase in VEGF concentra- tion. Conversely, in this same nude mouse model system, si- Group No. mice No. mice No. mice Ascites SV1 stable SKOV-3 cells were not tumorigenic and regressed that form with with volume after 3 weeks. One possibility, which would not exclude subcutaneous peritoneal ascites (ML) additional potential effects on proliferation and apoptosis, is tumors dissemination formation mean F SD that early-forming si-SV1 tumors were unable to sustain si-Luc 9/9 0/5 0/5 0.0 continued or increased growth secondary to deficient angio- si-KLF612/12 9/9 9/9 600.0 F 95.2 genic potential. Consistent with this hypothesis, si-SV1 stable cells grown in culture expressed significantly less VEGF (Fig. 5A). Suppression of VEGF expression by KLF6 is showed previously that, unlike KLF6, KLF6-SV1 does not potentially important because several clinical studies have directly up-regulate p21 or suppress cellular proliferation but shown the effectiveness of inhibiting VEGF-mediated angio- may antagonize KLF6 function in a dominant-negative manner genesis in treating ovarian cancer (3–6, 40). (20, 21). In ovarian tumors, KLF6-SV1 isoform expression was In contrast to effects of decreased KLF6-SV1, targeted increased in poorly differentiated, higher-staged serous tumors decreases in KLF6 expression result in a more aggressive and this increase correlated with increased KLF6 cytoplasmic cancer phenotype. Using a well-characterized genetically staining. Most notably, targeted siRNA-mediated KLF6-SV1 engineered model of dissemination, si-KLF6 SKOV-3 tumors knockdown in highly tumorigenic SKOV-3 cells blocked tumor were marked by increased i.p. dissemination and ascites growth. Therefore, potential treatment of ovarian cancer formation, mimicking the late-stage features of ovarian cancer. through targeted inhibition of KLF6-SV1 deserves further At the 6-week time point, only mice in the si-KLF6 group attention. developed multiple, visible peritoneal tumors with marked In addition to marked in vitro biological changes, in vivo VEGF-containing ascites. effects of KLF6 manipulation were also shown and correlated In summary, these findings show that the tumor suppressor with their effect on angiogenesis. Tumor angiogenesis is an gene KLF6 and its splice variant KLF6-SV1 play an important essential determinant for primary and metastatic tumor role in ovarian cancer development and progression. Ulti- growth (35, 36) and VEGF is known to act as a potent mately, we believe these findings have clinical relevance in angiogenic factor with additional important pathophysiologic identifying novel pathogenic associations for the KLF6 tumor consequences both in vitro and in vivo (37, 38). Over- suppressor family, defining interactions with clinically relevant expression of VEGF in ovarian cancer correlates with poor pathways, and highlighting down-regulation of KLF6-SV1 as a survival and metastatic spread (39). Therefore, it is particularly potential therapeutic target.

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Analisa DiFeo, Goutham Narla, Jennifer Hirshfeld, et al.

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