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• W2052970381 abstract "Emerging evidence suggests that the SAM pointed domain containing ETS transcription factor (SPDEF) plays a significant role in tumorigenesis in prostate, breast, colon, and ovarian cancer. However, there are no in vivo studies with respect to the role of SPDEF in tumor metastasis. The present study examined the effects of SPDEF on tumor cell metastasis using prostate tumor cells as a model. Utilizing two experimental metastasis models, we demonstrate that SPDEF inhibits cell migration and invasion in vitro and acts a tumor metastasis suppressor in vivo. Using stable expression of SPDEF in PC3-Luc cells and shRNA-mediated knockdown of SPDEF in LNCaP-Luc cells, we demonstrate for the first time that SPDEF diminished the ability of disseminated tumors cells to survive at secondary sites and establish micrometastases. These effects on tumor metastasis were not a result of the effect of SPDEF on cell growth as SPDEF expression had no effect on cell growth in vitro or subcutaneous tumor xenograft-growth in vivo. Transcriptional analysis of several genes associated with tumor metastasis, invasion, and the epithelial-mesenchymal transition demonstrated that SPDEF expression selectively down-regulated MMP9 and MMP13 in prostate cancer cells. Further analysis indicated that forced MMP9 or MMP13 expression rescued the invasive phenotype in SPDEF expressing PC3 cells in vitro, suggesting that the effects of SPDEF on tumor invasion are mediated, in part, through the suppression of MMP9 and MMP13 expression. These results demonstrate for the first time, in any system, that SPDEF functions as a tumor metastasis suppressor in vivo. Emerging evidence suggests that the SAM pointed domain containing ETS transcription factor (SPDEF) plays a significant role in tumorigenesis in prostate, breast, colon, and ovarian cancer. However, there are no in vivo studies with respect to the role of SPDEF in tumor metastasis. The present study examined the effects of SPDEF on tumor cell metastasis using prostate tumor cells as a model. Utilizing two experimental metastasis models, we demonstrate that SPDEF inhibits cell migration and invasion in vitro and acts a tumor metastasis suppressor in vivo. Using stable expression of SPDEF in PC3-Luc cells and shRNA-mediated knockdown of SPDEF in LNCaP-Luc cells, we demonstrate for the first time that SPDEF diminished the ability of disseminated tumors cells to survive at secondary sites and establish micrometastases. These effects on tumor metastasis were not a result of the effect of SPDEF on cell growth as SPDEF expression had no effect on cell growth in vitro or subcutaneous tumor xenograft-growth in vivo. Transcriptional analysis of several genes associated with tumor metastasis, invasion, and the epithelial-mesenchymal transition demonstrated that SPDEF expression selectively down-regulated MMP9 and MMP13 in prostate cancer cells. Further analysis indicated that forced MMP9 or MMP13 expression rescued the invasive phenotype in SPDEF expressing PC3 cells in vitro, suggesting that the effects of SPDEF on tumor invasion are mediated, in part, through the suppression of MMP9 and MMP13 expression. These results demonstrate for the first time, in any system, that SPDEF functions as a tumor metastasis suppressor in vivo. The development of a metastatic tumor continues to represent the most lethal and least treatable hallmark of cancer (1Hanahan D. Weinberg R.A. The hallmarks of cancer.Cell. 2000; 100: 57-70Abstract Full Text Full Text PDF PubMed Scopus (22336) Google Scholar). Metastases can occur years or decades after successful treatment of the primary tumor, which is due in part to tumor dormancy, the persistence of solitary cells at secondary sites, which can persist for extended periods of time in a secondary organ (2Chambers A.F. Groom A.C. MacDonald I.C. Dissemination and growth of cancer cells in metastatic sites.Nat. Rev. Cancer. 2002; 2: 563-572Crossref PubMed Scopus (3058) Google Scholar, 3Naumov G.N. MacDonald I.C. Chambers A.F. Groom A.C. Solitary cancer cells as a possible source of tumour dormancy?.Semin. Cancer Biol. 2001; 11: 271-276Crossref PubMed Scopus (101) Google Scholar, 4Naumov G.N. MacDonald I.C. Weinmeister P.M. Kerkvliet N. Nadkarni K.V. Wilson S.M. Morris V.L. Groom A.C. Chambers A.F. Persistence of solitary mammary carcinoma cells in a secondary site.Cancer Res. 2002; 62: 2162-2168PubMed Google Scholar). As the temporal and spatial order of biological events in metastasis continue to be elucidated in the laboratory setting; clinically, there remains no curative treatment for metastatic disease; therefore, continued experimental manipulation of the metastatic process to understand its biological mechanisms is warranted. ETS (E-twenty-six transformation-specific) transcription factors are involved in a multitude of normal and pathological cellular processes. Many ETS factors are deregulated and are thought to be key players in the generation of several types of cancer (5Sharrocks A.D. The ETS-domain transcription factor family.Nat. Rev. Mol. Cell Biol. 2001; 2: 827-837Crossref PubMed Scopus (816) Google Scholar). In prostate cancer, chromosomal rearrangements such as the ETS family member ERG-TMPRSS2 fusion represent an early event driving the development of prostate neoplasia progression. SPDEF (SAM pointed domain containing ETS transcription factor; also known as prostate-derived ETS factor) is the latest ETS family member discovered whose expression is limited to epithelial cells of the prostate, breast, lung, ovary, and colon. However, the role(s) SPDEF plays in tumorigenesis remains a subject of continued debate (reviewed in Refs. 6Steffan J.J. Koul H.K. Prostate-derived ETS factor (PDEF): A putative tumor metastasis suppressor.Cancer Lett. 2011; 310: 109-117Crossref PubMed Scopus (26) Google Scholar and 7Sood A.K. Kim H. Geradts J. PDEF in prostate cancer.Prostate. 2012; 72: 592-596Crossref PubMed Scopus (15) Google Scholar). Although several reports link SPDEF expression to tumor promotion, others demonstrate a role of tumor suppression in various models. In particular, Turner et al. (8Turner D.P. Moussa O. Sauane M. Fisher P.B. Watson D.K. Prostate-derived ETS factor is a mediator of metastatic potential through the inhibition of migration and invasion in breast cancer.Cancer Res. 2007; 67: 1618-1625Crossref PubMed Scopus (51) Google Scholar) demonstrated reduced invasive capacity of breast cancer cell lines when SPDEF was introduced and increased migration when SPDEF was knocked down via siRNA, whereas Gunawardane et al. (9Gunawardane R.N. Sgroi D.C. Wrobel C.N. Koh E. Daley G.Q. Brugge J.S. Novel role for PDEF in epithelial cell migration and invasion.Cancer Res. 2005; 65: 11572-11580Crossref PubMed Scopus (73) Google Scholar) demonstrated increased breast cell migration when SPDEF was overexpressed. Moreover, the single in vivo study published to date, using transformed mouse breast epithelial cells, found that SPDEF overexpression retarded both tumor incidence and growth in vivo (10Schaefer J.S. Sabherwal Y. Shi H.Y. Sriraman V. Richards J. Minella A. Turner D.P. Watson D.K. Zhang M. Transcriptional regulation of p21/CIP1 cell cycle inhibitor by PDEF controls cell proliferation and mammary tumor progression.J. Biol. Chem. 2010; 285: 11258-11269Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar); a mechanism dependent upon increased p21 expression. However, the precise role(s) of SPDEF in tumor growth and metastasis is not completely understood. Prostate cancer is the most frequently diagnosed, non-skin cancer in the United States and is the second leading cause of death in men. 2American Cancer Society (2012) Cancer Facts and Figures 2012, Atlanta, GA. Although conventional therapies produce a high cure rate for patients presenting with localized disease, there remains no curative treatment once the tumor has metastasized. Prostate tumor metastasis is the primary cause of mortality in prostate cancer patients. In addition, there are few, if any, reliable biomarker(s) that distinguish an indolent tumor, one which will respond well to conventional therapy from aggressive tumors, which require more aggressive therapy. In our previously published studies, we observed that SPDEF expression is reduced or lost in advanced stages of prostate cancer (12Johnson T.R. Koul S. Kumar B. Khandrika L. Venezia S. Maroni P.D. Meacham R.B. Koul H.K. Loss of PDEF, a prostate-derived Ets factor is associated with aggressive phenotype of prostate cancer: Regulation of MMP 9 by PDEF.Mol Cancer. 2010; 9: 148Crossref PubMed Scopus (48) Google Scholar). These studies also suggested that SPDEF expression suppressed an aggressive phenotype in prostate cancer. Our findings are in contrast to a report by Sood et al. (13Sood A.K. Saxena R. Groth J. Desouki M.M. Cheewakriangkrai C. Rodabaugh K.J. Kasyapa C.S. Geradts J. Expression characteristics of prostate-derived Ets factor support a role in breast and prostate cancer progression.Hum. Pathol. 2007; 38: 1628-1638Crossref PubMed Scopus (53) Google Scholar) but have been confirmed and even extended by two additional groups (14Tsujimoto Y. Nonomura N. Takayama H. Yomogida K. Nozawa M. Nishimura K. Okuyama A. Nozaki M. Aozasa K. Utility of immunohistochemical detection of prostate-specific Ets for the diagnosis of benign and malignant prostatic epithelial lesions.Int. J. Urol. 2002; 9: 167-172Crossref PubMed Scopus (17) Google Scholar, 15Ghadersohi A. Sharma S. Zhang S. Azrak R.G. Wilding G.E. Manjili M.H. Li F. Prostate-derived Ets transcription factor (PDEF) is a potential prognostic marker in patients with prostate cancer.Prostate. 2011; 71: 1178-1188Crossref PubMed Scopus (29) Google Scholar). In fact, Ghadersohi et al. (15Ghadersohi A. Sharma S. Zhang S. Azrak R.G. Wilding G.E. Manjili M.H. Li F. Prostate-derived Ets transcription factor (PDEF) is a potential prognostic marker in patients with prostate cancer.Prostate. 2011; 71: 1178-1188Crossref PubMed Scopus (29) Google Scholar) associated SPDEF expression with a favorable prognosis in localized prostate cancer. Taken together, based on these limited studies, the loss of SPDEF appears to be an indicator of aggressive prostate cancer. Experimental metastasis models do not recapitulate all steps of the metastatic cascade; however, the use of these models allows the study of the survival of disseminated tumor cells in the circulation, extravasation, and growth at secondary sites (16Bodenstine T.M. Welch D.R. Metastasis suppressors and the tumor microenvironment.Cancer Microenviron. 2008; 1: 1-11Crossref PubMed Scopus (40) Google Scholar, 17Alix-Panabières C. Riethdorf S. Pantel K. Circulating tumor cells and bone marrow micrometastasis.Clin. Cancer Res. 2008; 14: 5013-5021Crossref PubMed Scopus (197) Google Scholar). MMP9, 3The abbreviations used are: MMP9matrix metalloproteinase 9LucluciferaseVCvector controlSPDEF-KDSPDEF knockdownOEoverexpressed. also known as gelatinase B, is an extracellular matrix-degrading enzyme that is regarded as one of the classic metastasis-promoting genes (18van Kempen L.C. Coussens L.M. MMP9 potentiates pulmonary metastasis formation.Cancer Cell. 2002; 2: 251-252Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 19Deryugina E.I. Quigley J.P. Matrix metalloproteinases and tumor metastasis.Cancer Metastasis Rev. 2006; 25: 9-34Crossref PubMed Scopus (1638) Google Scholar) through either direct effects on substrate degradation, thereby promoting migration and invasion, or indirectly through prometastatic microenvironmental remodeling of the metastatic niche (19Deryugina E.I. Quigley J.P. Matrix metalloproteinases and tumor metastasis.Cancer Metastasis Rev. 2006; 25: 9-34Crossref PubMed Scopus (1638) Google Scholar, 20Itoh T. Tanioka M. Matsuda H. Nishimoto H. Yoshioka T. Suzuki R. Uehira M. Experimental metastasis is suppressed in MMP-9-deficient mice.Clin. Exp. Metastasis. 1999; 17: 177-181Crossref PubMed Scopus (283) Google Scholar, 21Hiratsuka S. Nakamura K. Iwai S. Murakami M. Itoh T. Kijima H. Shipley J.M. Senior R.M. Shibuya M. MMP9 induction by vascular endothelial growth factor receptor-1 is involved in lung-specific metastasis.Cancer Cell. 2002; 2: 289-300Abstract Full Text Full Text PDF PubMed Scopus (730) Google Scholar). MMP13, also known as collagenase-3, is known to degrade collagen and has been shown to be predominantly expressed at the invading front of tumor cells and is also produced by tumor-associated stromal fibroblasts (22Cazorla M. Hernández L. Nadal A. Balbín M. López J.M. Vizoso F. Fernández P.L. Iwata K. Cardesa A. López-Otín C. Campo E. Collagenase-3 expression is associated with advanced local invasion in human squamous cell carcinomas of the larynx.J. Pathol. 1998; 186: 144-150Crossref PubMed Scopus (0) Google Scholar). SPDEF and MMP9 levels have been found to inversely correlate in tissue samples, demonstrating that decreased SPDEF levels are associated with increased MMP9 expression in advanced disease (12Johnson T.R. Koul S. Kumar B. Khandrika L. Venezia S. Maroni P.D. Meacham R.B. Koul H.K. Loss of PDEF, a prostate-derived Ets factor is associated with aggressive phenotype of prostate cancer: Regulation of MMP 9 by PDEF.Mol Cancer. 2010; 9: 148Crossref PubMed Scopus (48) Google Scholar). Numerous studies have shown a role for SPDEF in cell migration and invasion in vitro using cell lines of multiple tissue origin (6Steffan J.J. Koul H.K. Prostate-derived ETS factor (PDEF): A putative tumor metastasis suppressor.Cancer Lett. 2011; 310: 109-117Crossref PubMed Scopus (26) Google Scholar, 12Johnson T.R. Koul S. Kumar B. Khandrika L. Venezia S. Maroni P.D. Meacham R.B. Koul H.K. Loss of PDEF, a prostate-derived Ets factor is associated with aggressive phenotype of prostate cancer: Regulation of MMP 9 by PDEF.Mol Cancer. 2010; 9: 148Crossref PubMed Scopus (48) Google Scholar, 23Feldman R.J. Sementchenko V.I. Gayed M. Fraig M.M. Watson D.K. Pdef expression in human breast cancer is correlated with invasive potential and altered gene expression.Cancer Res. 2003; 63: 4626-4631PubMed Google Scholar, 24Turner D.P. Findlay V.J. Moussa O. Semenchenko V.I. Watson P.M. LaRue A.C. Desouki M.M. Fraig M. Watson D.K. Mechanisms and functional consequences of PDEF protein expression loss during prostate cancer progression.Prostate. 2011; 71: 1723-1735Crossref PubMed Scopus (49) Google Scholar); however, to date, no in vivo studies have been performed investigating the effect of SPDEF on tumor metastasis. In the present study, we evaluated the functions of SPDEF using luciferase-expressing prostate cancer cell lines (PC3-Luc and LNCaP-Luc). Results presented herein demonstrate for the first time that stable expression of SPDEF in PC3-Luc cells decreased, whereas stable knockdown of SPDEF in LNCaP-Luc prostate tumor cells increased, the ability of these cells to survive at metastatic sites. Finally, we found that SPDEF-mediated down-regulation of MMP9 and MMP13 represents a significant mechanism of action in that MMP9 or MMP13 overexpression is sufficient to overcome the inhibitory effects of SPDEF expression on tumor cell invasion. To the best of our knowledge, this is the first study, in any model, demonstrating a tumor metastasis suppressor function of SPDEF in vivo. matrix metalloproteinase 9 luciferase vector control SPDEF knockdown overexpressed. Cells were cultured, and the cloning of SPDEF and creation of stable SPDEF expressing and vector control PC3 cell lines were described previously (12Johnson T.R. Koul S. Kumar B. Khandrika L. Venezia S. Maroni P.D. Meacham R.B. Koul H.K. Loss of PDEF, a prostate-derived Ets factor is associated with aggressive phenotype of prostate cancer: Regulation of MMP 9 by PDEF.Mol Cancer. 2010; 9: 148Crossref PubMed Scopus (48) Google Scholar). To engineer luciferase expression, 6 × 106 cells were seeded in six-well plates. The following day, cells were infected with a luciferase delivering lentivirus virus with the addition of 8 μg/ml polybrene. Clonal selection was performed, and clones were screened for luciferase expression. shRNA constructs were obtained from the University of Colorado Functional Genomics core facility in the pLKO1 backbone. The plasmid along with a packaging and envelop plasmid (Addgene) was transfected into 8 × 105 293T cells using Effectene (Qiagen) according to the manufacturer's instructions. After 24 h, the medium was changed (20 mm HEPES, 30% FBS/DMEM, 2 mm sodium butyrate). After 24 h, the medium was collected, filtered through 0.45-μm filters, and flash frozen. Cells were infected with lentiviral particles at a multiplicity of infection of 1. Two shRNA sequences were used for SPDEF. Clones were picked from cell pools selected via puromycin and were tested for knockdown efficiency via Western blot. The scrambled (Scr) shRNA vector contains four base pair mismatches within the short hairpin sequence to any known human gene, serving as a negative control. Each SPDEF-specific shRNA construct had the same phenotypic effect (data not shown), thus eliminating off-target effects of the shRNA and between cell clones. Multiple cell clones from both shRNAs demonstrated similar results. The following antibodies were used: p21 (1:1000) mouse monoclonal, p27 (1:1000) rabbit polyclonal, and CDK2 (1:1000) rabbit monoclonal were purchased from Cell Signaling; PDEF N-14 (1:250) goat polyclonal from Santa Cruz Biotechnology, and actin (1:1000) rabbit polyclonal and anti-FLAG were purchased from Sigma. These were performed as described previously (25Steffan J.J. Snider J.L. Skalli O. Welbourne T. Cardelli J.A. Na+/H+ Exchangers and RhoA regulate acidic extracellular pH-induced lysosome trafficking in prostate cancer cells.Traffic. 2009; 10: 737-753Crossref PubMed Scopus (80) Google Scholar). MTT assays were performed as described previously (26Kumar B. Koul S. Khandrika L. Meacham R.B. Koul H.K. Oxidative stress is inherent in prostate cancer cells and is required for aggressive phenotype.Cancer Res. 2008; 68: 1777-1785Crossref PubMed Scopus (543) Google Scholar). Wound healing assays were performed as described previously (12Johnson T.R. Koul S. Kumar B. Khandrika L. Venezia S. Maroni P.D. Meacham R.B. Koul H.K. Loss of PDEF, a prostate-derived Ets factor is associated with aggressive phenotype of prostate cancer: Regulation of MMP 9 by PDEF.Mol Cancer. 2010; 9: 148Crossref PubMed Scopus (48) Google Scholar). A TrueClone MMP9 or MMP13 expression plasmid was purchased from OriGene, and 0.4 μg of DNA was transfected using Effectene (Qiagen) according to the manufacturer's instructions. Cells were transfected in suspension and seeded into Matrigel-coated Boyden chambers as described below. RNA was isolated per manufacturer's instructions (Qiagen; RNeasy). cDNA was synthesized using cDNA iScript (Bio-Rad) per the manufacturer's instructions. PCR was then performed using standard Taq polymerase (Fermentas) with the following cycles: 95° C for 4 min; (95° C for 30 s, annealing temperature of 1 min, 72° C for 1 min) ×38 cycles; 72° C for 10 min. Annealing temperatures were 2° C lower than the primer melting temperatures found in Table 1. Primer sequences can also be found in Table 1.TABLE 1RT-PCR primer sequencesGeneRT-PCR primer sequencesForwardTemperature (°C)ReverseTemperature (°C)Cyclin B5′-TTGATACTGCCTCTCCAAGCCCAA-3′60.35′-TTGGTCTGACTGCTTGCTCTTCCT-3′60.3Cyclin D5′-CCTTTGGTGCCAACTGGTGTTTGA-3′60.35′-TCAGATGACTCTGGGAAACGCCAA-3′60.3Cyclin E5′-TGCAGAGCTGTTGGATCTCTGTGT-3′60.35′-ACAACATGGCTTTCTTTGCGCGGG-3′60.3CDK25′-AGCCAGAAACAAGTTGACGGGAGA-3′60.55′-AAGAGGAATGCCAGTGAGAGCAGA-3′60.0p275′-AGTCCATTTGATCAGCGGAGACTCG-3′60.45′-TCGCACGTTTGACATCTTTCTCCC-3′59.5p215′-TTCGACTTTGTCACCGAGACACCA-3′60.25′-AGGCACAAGGGTACAAGACAGTGA-3′60.1VASP5′-GTAAGAGTAACACTGTAGCCGCCA-3′58.55′-ATCATAAAGCATCACAGTGGCCCG-3′59.6Survivin5′-GAGGCTGGCTTCATCCACTG-3′58.25′-CAGCTGCTCGATGGCACGGC-3′64.1Snail5′-TGCCAATGCTCATCTGGGACTCT-3′60.55′-GCCTCCAAGGAAGAGACTGAAGTA-3′57.9MMP95′-TACCACCTCGAACTTTGACAGCGA-3′60.15′-AAAGGCACAGTAGTGGCCGTAGAA-3′60.3Slug5′-ACTACAGTCCAAGCTTTCAGACCC-3′58.65′-CCGCAGATCTTGCAAACACAAGGT-3′60.2MMP135′-AACATCCAAAAACGCCAGAC-3′53.95′-GGAAGTTCTGGCCAAAATGA-3′53.3SPDEF5′-CAGGTGAAGTCCGCTCTTTC-3′55.75′-AATGTGCAGAAGTGGCTCCT-3′56.9IL-65′-TACCCCCAGGAGAAGATTCC-3′55.75′-AAAGCTGCGCAGAATGAGAT-3′55.0CXCR45′-TGGCCTTATCCTGCCTGGTATTGT-3′60.25′-TGGCTCCAAGGAAAGCATAGAGGA-3′59.9GAPDH5′-AAGGTCGGAGTCAACGGATTTGGT-3′60.55′-AGTGATGGCATGGACTGTGGTCAT-3′60.5 Open table in a new tab Densitometry was performed using ImageJ software. All mRNA bands were compared with GAPDH bands. Immunohistochemistry was performed as described previously (12Johnson T.R. Koul S. Kumar B. Khandrika L. Venezia S. Maroni P.D. Meacham R.B. Koul H.K. Loss of PDEF, a prostate-derived Ets factor is associated with aggressive phenotype of prostate cancer: Regulation of MMP 9 by PDEF.Mol Cancer. 2010; 9: 148Crossref PubMed Scopus (48) Google Scholar). The invasion assay was performed as described previously (27Steffan J.J. Cardelli J.A. Thiazolidinediones induce Rab7-RILP-MAPK-dependent juxtanuclear lysosome aggregation and reduce tumor cell invasion.Traffic. 2010; 11: 274-286Crossref PubMed Scopus (25) Google Scholar), with the exception of 10% serum containing medium in the bottom chamber. 1% serum conditions were used in the top chamber in cells that were transfected with MMP9 or MMP13 constructs. Cells were grown in eight-well chamber slides coated with Matrigel. After cell seeding, 4% Matrigel was added to the top of the cells. Medium was changed every other day for 10–15 days. See Ref. 12Johnson T.R. Koul S. Kumar B. Khandrika L. Venezia S. Maroni P.D. Meacham R.B. Koul H.K. Loss of PDEF, a prostate-derived Ets factor is associated with aggressive phenotype of prostate cancer: Regulation of MMP 9 by PDEF.Mol Cancer. 2010; 9: 148Crossref PubMed Scopus (48) Google Scholar for more details. Six- to 8-week-old male nude mice (Taconic NCRNU) were injected subcutaneous with 1.5 × 106 cells in 100 μl PBS/Matrigel (50:50) subcutaneously (n = 5 VC; n = 5 SPDEF OE). Tumors became measurable by day 22 post-implantation and were measured with calipers twice per week. Tumor volumes were calculated by the following equation: volume = π/6 × length × width2. Mice were euthanized at 42 days post-implantation, and tumors were surgically removed and fixed in 10% formaldehyde or flash frozen. All animal experiments were performed in accordance with guidelines set by the University of Colorado Institutional Animal Care and Use Committee. Six- to 8-week-old male nude mice (Taconic-NCR-nu) were injected via either intracardiac or tail vein with 1 × 106 cells in 100 μl of PBS. An insulin needle was used for the tail vein injections, whereas a 26-gauge needle was used for the intracardiac injection. Mice were imaged for luciferase signal by an intraperitoneal injection of luciferin (Caliper Life Sciences) (15 μg in 100 μl of PBS) every other week for 12 weeks using a Xenogen system. GraphPad Software (Prism, version 3.0) was utilized to perform all statistics. Mann-Whitney T-tests were performed to indicate statistical significance. All error bars display the S.E. The expression levels of SPDEF varies among prostate tumor cell lines (12Johnson T.R. Koul S. Kumar B. Khandrika L. Venezia S. Maroni P.D. Meacham R.B. Koul H.K. Loss of PDEF, a prostate-derived Ets factor is associated with aggressive phenotype of prostate cancer: Regulation of MMP 9 by PDEF.Mol Cancer. 2010; 9: 148Crossref PubMed Scopus (48) Google Scholar). PC3 prostate tumor cells express low to undetectable levels of SPDEF protein and are known to have high metastatic potential in nude mouse models, whereas LNCaP tumor cells express relatively high levels of SPDEF and lack metastatic potential in vivo. Stable expression of FLAG-tagged SPDEF in PC3 cells was achieved as described previously (12Johnson T.R. Koul S. Kumar B. Khandrika L. Venezia S. Maroni P.D. Meacham R.B. Koul H.K. Loss of PDEF, a prostate-derived Ets factor is associated with aggressive phenotype of prostate cancer: Regulation of MMP 9 by PDEF.Mol Cancer. 2010; 9: 148Crossref PubMed Scopus (48) Google Scholar). SPDEF-targeted shRNA in a lentiviral vector was used to create stable knockdowns of SPDEF in LNCaP cells, whereas vector control LNCaP cells received Scr-shRNA via a lentiviral vector. All four cell lines VC-PC3 cells, FLAG-tagged SPDEF expressing (SPDEF-PC3) cells, Scr shRNA LNCaP cells and SPDEF knockdown (SPDEF-KD) LNCaP cells were stably transfected with luciferase using a lentiviral vector to generate VC-PC3-Luc, SPDEF-PC3-Luc, Scr-LNCaP-Luc, and SPDEF-KD-LNCaP-Luc cells, respectively. Luciferase was visualized via bioluminescence imaging, and no significant difference in luciferase expression was detected between the cells lines (Fig. 1A). The expression and knockdown of SPDEF was confirmed in luciferase-expressing cells as evidenced via an anti-SPDEF and/or anti-FLAG Western blot (Fig. 1B). For these studies, we used Luc-tagged VC-PC3-Luc, SPDEF-PC3-Luc, Scr-LNCaP-Luc, and SPDEF-KD-LNCaP-Luc cells. We found that stable expression of SPDEF expression resulted in decreased cell motility as SPDEF-PC3-Luc cells demonstrated significantly decreased motility in a directional migration assay as compared with VC-PC3-Luc cells. In addition, SPDEF-KD-LNCaP-Luc cells were more motile compared with Scr-LNCaP-Luc control cells, suggesting that SPDEF expression results in decreased cell motility (Fig. 1C). Moreover, modulation of SPDEF expression affected cell invasion using a Matrigel-coated Boyden chamber assay. We observed that SPDEF-PC3-Luc cells had significantly decreased ability to invade as compared with VC-PC3-Luc cells, whereas SPDEF-KD-LNCaP-Luc cells were more invasive as compared with Scr-LNCaP-Luc control cells, suggesting that SPDEF expression was an impediment to cell invasion (Fig. 1D). Lastly, stable expression of SPDEF appeared to reverse the aggressive pattern of growth of these cells in three-dimensional cell culture (Fig. 1E), whereas SPDEF knockdown in LNCaP-Luc cells caused a more subtle increase in the aggressive appearance of LNCaP-Luc cells in a three-dimensional culture (Fig. 1E). These results are in agreement with numerous studies demonstrating the relationship between phenotypic three-dimensional spheroid formation with cell migration and invasion (28Findlay V.J. Turner D.P. Yordy J.S. McCarragher B. Shriver M.R. Szalai G. Watson P.M. Larue A.C. Moussa O. Watson D.K. Prostate-derived ETS factor regulates epithelial-to-mesenchymal transition through both SLUG-dependent and independent mechanisms.Genes Cancer. 2011; 2: 120-129Crossref PubMed Scopus (16) Google Scholar, 29Moss N.M. Liu Y. Johnson J.J. Debiase P. Jones J. Hudson L.G. Munshi H.G. Stack M.S. Epidermal growth factor receptor-mediated membrane type 1 matrix metalloproteinase endocytosis regulates the transition between invasive versus expansive growth of ovarian carcinoma cells in three-dimensional collagen.Mol. Cancer Res. 2009; 7: 809-820Crossref PubMed Scopus (32) Google Scholar). Thus, our results suggest that SPDEF modulation effects cell migration and invasion, namely when SPDEF expression is high, cells are less motile and invasive, and conversely, when SPDEF expression is low, cells demonstrate increased motility and invasion. Because SPDEF expression decreased tumor cell migration and invasion in vitro, the hypothesis that SPDEF expression effects tumor metastasis was analyzed. Two experimental metastasis models were used. First, VC-PC3-Luc cells or SPDEF-PC3-Luc cells were injected into either the tail vein or into the arterial circulation (intracardiac injection) of nude mice. Mice were imaged 1 week following the injection for up to 12 weeks (Fig. 2, A and B). Both the VC-PC3-Luc cells and SPDEF-PC3-Luc cells were found to have circulated well, and no significant difference in the metastatic seeding ability was detected between these two cell lines at 1 week following injections. However, two mice in the intracardiac model (numbered two and five in Fig. 2B) had tumor cell growth near the heart. These two mice were eliminated from further imaging and all quantitation to quantitate only those cells that were successfully disseminated in to circulation. At the end of 12 weeks, in vivo bioluminescent imaging (Fig. 2, A and B) and quantitation (Fig. 2, C and D) revealed that the SPDEF-PC3-Luc cells significantly failed to survive and develop micrometastases as compared with the VC-PC3-Luc cells, suggesting that SPDEF expression reduces disseminated cell survival in both the tail vein and intracardiac metastasis models. In parallel experiments, we utilized the SPDEF-KD-LNCaP-Luc cells and Scr-LNCaP-Luc cells and injected them into either the tail vein or into the arterial circulation (intracardiac injection) of nude mice. Both the SPDEF-KD-LNCaP-Luc cells and Scr-LNCaP-Luc cells were found to have circulated well, and no significant difference in the metastatic seeding ability was detected between these two cell lines at week 1. At the end of 12 weeks, in vivo bioluminescent imaging (Fig. 3, A and B) and quantitation (Fig. 3, C and D) revealed that the SPDEF-KD-LNCaP-Luc cells survived significantly better and developed micrometastases compared with the Scr-LNCaP-Luc cells, suggesting that SPDEF knockdown increases survival of disseminated cell in both the tail vein and intracardiac metastasis models. Taken together, these results demonstrate the ability of SPDEF to inhibit the ability of circulating tumor cells to develop into successful metastasis. To test whether or not SPDEF modulated tumor metastasis in vivo by altering tumor cell growth, we evaluated the effects of differential SPDEF expression on cell growth in vitro. For these studies, we compared growth curves of VC-PC3-Luc cells and SPDEF-PC3-Luc cell" @default.
• W2052970381 created "2016-06-24" @default.
• W2052970381 creator A5033528355 @default.
• W2052970381 creator A5037312238 @default.
• W2052970381 creator A5053019599 @default.
• W2052970381 creator A5067910048 @default.
• W2052970381 date "2012-08-01" @default.
• W2052970381 modified "2023-10-15" @default.
• W2052970381 title "The Transcription Factor SPDEF Suppresses Prostate Tumor Metastasis" @default.
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