Oncogene (2007) 26, 1739–1747 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE Transcriptional regulation of KiSS-1 expression in metastatic melanoma byspecificityprotein-1 and its coactivator DRIP-130

DC Mitchell1, LJ Stafford1,DLi1, M Bar-Eli2 and M Liu1

1Institute of Biosciences and Technology, and Department of Molecular and Cellular Medicine, Texas A&M University System Health Science Center, Houston, TX, USA and 2Department of Cancer Biology, University of Texas MD Anderson Cancer Center, Houston, TX, USA

Loss of the metastasis suppressor gene, KiSS-1 has been Several , known as metastasis suppressors, have stronglycorrelated to the progression of metastases in recently been identified which inhibit metastasis, and numerous types of cancers. The mechanism through which little is known regarding the mechanisms by which they KiSS-1 is lost during metastasis, however, is still not act or how their expression is regulated in both normal completelyknown. Previous studies have shown that and tumorigenic tissues (Steeg et al., 2003). KiSS-1, is a genetic material on human 6q16.3–q23 is precursor for secreted peptide ligands for the G-protein essential for KiSS-1 expression in normal tissues. coupled receptor, hGRP54 (Kotani et al., 2001; Muir Additionally, microcell-mediated transfer of this chromo- et al., 2001; Ohtaki et al., 2001; Stafford et al., 2002). some in cancerous tissue results in rescued expression of Microcell-mediated transfer of highly metastatic me- KiSS-1 and reduced metastatic phenotype. Here, we show lanoma was initially used to identify KiSS-1 as a that loss of Sp1-coactivator protein DRIP-130, which is metastasis suppressor gene, as it was shown to reduce encoded byhuman chromosome 6q16.3–q23, results in invasive and migratory properties without affecting reduced KiSS-1 promoter activation in highlymalignant tumorigenicity (Lee and Welch, 1997b). Similar to that melanoma cells. Co-expression of Sp1 and DRIP-130 not of other known metastasis suppressors, the expression of onlyrescues KiSS-1 expression, but also induces an KiSS-1 is commonly found to be inversely correlated to inhibition of the invasive and migratorybehavior in highly the degree of cancer metastasis. Consequently, loss of metastatic melanoma cells, similar to the overexpression KiSS-1 expression has been shown to be a good of KiSS-1 metastasis suppressor gene in those cells. indicator for the progression of several types of cancers Furthermore, we demonstrate that KiSS-1 expression is (Lee and Welch, 1997b; Sanchez-Carbayo et al., 2003; regulated bySp1 elements within the first 100-bp region of Ikeguchi et al., 2004; Masui et al., 2004; Jiang et al., the KiSS-1 promoter and that targeted deletion of a single 2005). Additionally, overexpression or treatment with GC-rich region spanning À93 to À58 interrupts Sp1- and KiSS-1 peptide, reduces invasive and migratory proper- DRIP-130-modulated transcriptional control of KiSS-1 ties of cancer cells in vitro (Lee and Welch, 1997a, b; expression. Our results thus suggest that DRIP-130 is a Ohtaki et al., 2001; Stafford et al., 2002; Masui et al., keyregulator in KiSS-1 transactivation in normal tissue, 2004). Several labs have shown that deletion of 6q16.3– and that the loss of DRIP-130 expression, as a result q23 and loss of heterozygosity correlate with increased of the gross loss of human chromosome 6q16.3–q23, metastasis and decreased KiSS-1 expression, suggesting provokes increased tumor metastasis. that KiSS-1 may be regulated by genes encoded within Oncogene (2007) 26, 1739–1747. doi:10.1038/sj.onc.1209963; that region (Miele et al., 2000; Shirasaki et al., 2001). published online 11 September 2006 The objective of this study was thus to identify the upstream regulator(s) of KiSS-1 which localize to Keywords: KiSS1; GPR54; Sp1; CRSP3; cancer meta- chromosome 6q16.3–q23. stasis DRIP-130, also known as cofactor required for Sp1 transcriptional activation (CRSP3), is one of 15 subunits which compose the transcriptional cofactor complex CRSP/, and is required for transcriptional Introduction activation by Sp1 (Ryu and Tjian, 1999; Ryu et al., 1999; Taatjes and Tjian, 2004). Previous research Metastasis is an involved process in which cancer cells suggests DRIP-130 itself acts as a metastasis suppressor, invade new tissues and develop as secondary cancers. and that its expression correlates directly to the expression of other metastasis suppressors in clinical Correspondence: Dr M Liu, Institute of Biosciences and Technology, samples of melanoma, thus making DRIP-130 a likely Texas A&M University System Health Science Center, 2121 W. and interesting candidate regulatory factor of KiSS-1 Holcombe Blvd., Houston, TX 77030, USA. E-mail: [email protected] (Goldberg et al., 2003). Here, we report that re- Received 8 May 2006; revised 5 July 2006; accepted 6 July 2006; introduction of DRIP-130 in highly metastatic mela- published online 11 September 2006 noma cells induces higher KiSS-1 expression, and that Regulation of KiSS-1 expression in melanoma by Sp1 and DRIP-130 DC Mitchell et al 1740 coexpression of Sp1 and DRIP-130 decreases invasive and migratory properties of metastatic melanoma cells. Furthermore, we demonstrate that KiSS-1 expression is modulated by Sp1 elements within the first 100-bp region of the KiSS-1 promoter and that targeted deletion of a single GC-rich region spanning À93 to À58 of the promoter interrupts Sp1- and DRIP-130- modulated transcriptional control of KiSS-1 expression. Therefore, our data provides a regulatory mechanism for the loss of KiSS-1 gene expression commonly seen in metastatic melanomas and correlates it with the gross loss of genetic material on human chromosome 6q16.3– q23 that leads to heightened tumor metastasis.

Results

Identification of the regulatory region of KiSS-1 promoter by Sp1 and its coactivator DRIP-130in melanoma The full-length human KiSS-1 promoter has a high GC- content and contains multiple putative Sp1 sites, with more than 10 overlapping putative Sp1 sites in the first 100 bases alone (see Figure 1a). Recent evidence has revealed that the transcription factor, Sp1, requires the presence of other cofactors, including the CRSP complex, for proper assembly on Sp1-modulated pro- moters. (Ryu et al., 1999). To determine whether the transcriptional effect of Sp1 on KiSS-1 expression in melanoma is modulated by the Sp1 co-activator, DRIP- 130, we examined the effects of Sp1 and DRIP-130 on activation of KiSS-1 promoter using a series of KiSS-1 promoter-bearing luciferase constructs (see Figure 1a). Luciferase assays were carried out as described pre- viously, and normalized using b-galactosidase levels Figure 1 The 100 bp region of human KiSS-1 promoter contains (Mitchell et al., 2006). As shown in Figure 1b, whereas multiple overlapping Sp1 sites regulated by DRIP-130 and Sp1. single transfections of DRIP-130 and Sp1 had no effect, (a) Schematic representation of human KiSS-1 promoter-driver luciferase constructs contain 35 bp cluster of overlapping putative co-expression of both Sp1 and DRIP-130 resulted in a Sp1 transcription factor binding elements (SP1 CLUSTER) in six- eightfold increase in the highly metastatic melanoma addition to two smaller individual putative Sp1 sites (black boxes). cell line, WM2664 (Figure 1b). To determine the discrete (b) Sp1 and DRIP-130 activate KiSS-1 promoter-luciferase region modulating Sp1 and DRIP-130 response, luci- constructs, including the 1 Kb, the 150 bp, and the first 100 bp preceding the start site. (c) Luciferase assays in both metastatic ferase assays using serial truncations of the KiSS-1 melanoma (WM2664 and A375SM) and breast cancer cells (T47D promoter sequence were performed. Nearly the same and MDA-435) show a synergistic effect of Sp1 and DRIP-130 on fold increase in KiSS-1 transactivation was observed transcriptional activation of the 100 bp KiSS-1 promoter specific to using the 1000-, 150- and the 100- sequence of melanoma cells. the KiSS-1 promoter, suggesting that the region modulating KiSS-1 transactivation was located within the first 100 bases (Figure 1b). KiSS-1 levels in highly metastatic melanoma correlates To examine whether the transcriptional effects of Sp1 directly to the expression of DRIP-130mRNA and and DRIP-130 were tissue/cell specific, we measured protein activation of the KiSS-1 promoter by Sp1 and DRIP- To determine whether a correlation existed between the 130 in four different metastatic cancer cell lines, two expression of KiSS-1, Sp1 and DRIP-130 in melanoma, melanoma (WM2664 and A375SM) and two breast we examined their expression in melanoma cell lines of cancer cell lines (T47D and MDA-435). As shown different metastatic capacities (Figure 2a and b). Owing Figure 1c, co-transfection of Sp1 and DRIP-130 greatly to the lack of a specific and effective KiSS-1 antibody, enhanced activation in metastatic melanoma cells, KiSS-1 expression was quantitated using reverse tran- however, only slight activation was observed when scription-polymerase chain reaction (RT–PCR) and co-transfection was done using the two highly meta- real-time PCR, and then normalized to the expression static breast cancer cell lines (Figure 1c), suggesting that levels of b-actin and glyceraldehyde-3-phosphate dehy- KiSS-1 transactivation by Sp1 and DRIP-130 may be drogenase, respectively in Figure 2a and b. Results tissue-specific (Figure 1c). showed that the more metastatic cell lines, A375SM and

Oncogene Regulation of KiSS-1 expression in melanoma by Sp1 and DRIP-130 DC Mitchell et al 1741 compared to the actin loading control. To determine whether reduced expression of DRIP-130 in melanoma was responsible in part for significantly decreased KiSS-1 levels found in the more metastatic WM2664 and A375SM melanoma cell lines, Sp1 and DRIP-130 were singly transfected into these cells, which were then lysed and analysed for KiSS-1 expression using RT–PCR. As no significant increase in KiSS-1 messen- ger RNA (mRNA) levels was seen (results not shown), cells were then co-transfected with both Sp1 and DRIP- 130. Results demonstrated that co-transfection induced KiSS-1 expression levels in melanoma (Figure 2c, middle, co-transfected) as compared to the untrans- fected cells (Figure 2c, top). This data suggests that overexpression of both Sp1 and DRIP-130 is required for increased KiSS-1 expression in metastatic melanoma cell lines.

Determination of the KiSS-1 promoter region responsive to transcriptional upregulation by DRIP-130and Sp1 To demonstrate the importance of the first 100 bases in mediating the effects of Sp1 and DRIP-130, we measured the levels of luciferase induction by constructs driven by just the first 100 bp of the KiSS-1 promoter or by a promoter construct with targeted deletion of this region. Results showed that this 100 bp region was sufficient for the transactivation mediated by DRIP-130 and Sp1 (Figure 3a), and that targeted deletion resulted in the loss of response to Sp1 and DRIP-130 (Figure 3a, last three columns). Additionally, these results suggest that whereas many putative Sp1 sites are located throughout the full-length human KiSS-1 promoter, the transcriptional effects of DRIP-130 and Sp1 may be confined to the first hundred bases of the promoter. To further support the significance of Sp1 binding elements localized within the 100 bp region proximal to Figure 2 Reduced expression of DRIP-130 in highly metastatic the KiSS-1 start site, Electrophoretic mobility shift assay melanoma cells correlates with the expression level of KiSS-1 gene. (EMSA) was performed to determine whether these sites (a) Western blot analysis of DRIP-130 and Sp1 was compared to were physically capable of binding the Sp1 transcription KiSS-1 RT–PCR analysis in both non-metastatic (MeWo and SB2) factor. Within the first hundred base pairs of the human and metastatic (WM2664 and A375SM) melanoma cells. Reduced DRIP-130 expression seen in metastatic melanoma correlated KiSS-1 promoter sequence, a single GC-rich region directly with reduced mRNA levels of KiSS-1. Sp1 levels remained spanning À93 to À58 and consisting of approximately relatively unchanged between cell lines examined. Actin controls ten overlapping putative Sp1 elements stood out as the for Western analysis and b-actin controls for RT–PCR analysis likely candidate for modulating DRIP-130 and Sp1 were carried out to ensure equal loading of samples. (b) Real-time PCR analysis of KiSS-1 expression in non-metastatic (MeWo) and upregulation and was subsequently the first tested for metastatic cells (WM2664 and A375SM) melanoma cells. Expres- Sp1 binding. WM2664 cells, which were transfected to sion of KiSS-1 is dramatically reduced in metastatic melanoma overexpress DRIP-130 and Sp1, were lysed and incu- cells. (c) Co-transfection of WM-2664 and A375SM cells with bated with a radiolabeled probe spanning fifty bases of DRIP-130 and Sp1 resulted in higher KiSS-1 mRNA expression the promoter sequence including the candidate GC-rich (Muir et al., 2001) in RT–PCR analysis as compared with the untransfected melanoma lines (top). region. The resulting patterns of bands revealed that Sp1 formed a DNA–protein complex (Figure 3b, lane 2). Incubation of an unlabeled probe competed for protein WM2664, demonstrated considerably less KiSS-1 and binding, and resulted in a reduction of DNA–protein DRIP-130 expression, as compared to the lesser meta- complex and band intensity (Figure 3b, lane 3). Only the static SB2 and MeWo cell lines (Figure 2a). Further- Sp1-specific antibody was capable of super shifting the more, real-time PCR analysis demonstrated dramatic DNA–protein complex (Figure 3b, lane 5) whereas the decrease in KiSS-1 expression in highly metastatic cell control anti-immunoglobulin (Ig)G antibody had no lines, WM2664 and A375SM (Figure 2b). Although a effect (Figure 3b, lane 4). The negative control in which slight increase in Sp1 expression was seen in the SB2 labeled probe is seen in the absence of nuclear extract is line, Sp1 expression remained relatively constant, as shown in Figure 3b, lane 1. EMSA results using Sp1/

Oncogene Regulation of KiSS-1 expression in melanoma by Sp1 and DRIP-130 DC Mitchell et al 1742 chromatin bound 100-bp fragment of the KiSS-1 promoter was examined. Chromatin immunoprecipita- tion (ChIP) analysis in which sheared DNA isolated from DRIP-130/Sp1 co-transfected WM2664 melanoma cells was immunoprecipitated overnight using antibo- dies specific to DRIP-130, Sp1 and IgG. primers, designed to overlap the Sp1-rich cluster between -1 and -150, were used to amplify the fragment of the immunoprecipitated chromatin (Figure 3c). Results showed that the Sp1 antibody was able to immunopre- cipitate the first 150-bp of the chromatin-bound KiSS-1 promoter (Figure 3d, lane 4). However, anti-IgG, the negative control, did not precipitate the KiSS-1 promo- ter and thus did not form PCR product (Figure 3d, lane 3). Because ChIP using endogenous levels of protein were inconclusive, we chose to use lysates in which both Sp1 and DRIP-130 were overexpressed, resulting in an additive effect of DNA–protein interac- tion, by which we could demonstrate specific Sp1 interaction with the KiSS-1 promoter. Controls included the PCR products using both the non-immunoprecipi- tated input (lane 2) and a vector containing the full-length KiSS-1 promoter sequence (lane 1). Further- more, the antibody to DRIP-130 immunoprecipitated this region of the KiSS-1 promoter (Figure 3d, lane 5). Previous incubation of the DRIP-130-specific antibody in EMSA assays showed no conclusive DNA–protein Figure 3 Sp1 cluster in the first 100 bp of KiSS-1 promoter is interaction (data not shown), however, such seemingly essential for modulating DRIP-130/Sp1-mediated activity. inconsistent findings may likely result from even very (a) Targeted deletion of 100 bp region of KiSS-1 promoter containing Sp1 cluster loses its response to activation by Sp1 and small differences in the antibody or protein binding DRIP-130. Expression of a 900 bp KiSS-1 promoter-driven conditions in EMSA and ChIP assays. Generally, luciferase construct lacking the first 100 bp (D100 bp KiSS-1) however, ChIPP assays are considered more sensitive eliminates DRIP-130/Sp1 modulation, when expressed in malig- and ChiP assays using A375SM cells were consistent nant melanoma WM2664. (b) EMSA reveals Sp1 interaction with the Sp1 cluster (lane 5). This DNA–protein interaction is specific with WM264 results. From our ChIP assays, we for Sp1 (lane 5), as anti-IgG was incapable of supershifting the conclude that DRIP-130 exists in a protein complex bands (lane 4). Additionally, competition for protein binding was that binds to the Sp1 cluster between -93 and -58 in the shown using unlabeled probe (Â 100 CC; lane 3). Probe controls chromatin-bound form. in the presence (lane 2) and absence (lane 1) are also shown. (c) Primers spanning the first 156 bp of the KiSS-1 promoter were designed for CHiP to determine if Sp1 and/or DRIP-130 antibodies could precipitate the Sp1 cluster-containing portion of the Co-transfection of metastatic melanoma cell lines with chromatin-bound KiSS-1 promoter. (d) CHiP demonstrated DRIP-130and Sp1 results in reduced migratory and specific binding of Sp1 and DRIP-130 proteins (lanes 4 and 5, invasive properties in vitro respectively) to the chromatin-bound KiSS-1 promoter in WM2664 cells. Precipitation using an IgG-specific antibody was used as a KiSS-1 is known to inhibit migration and invasion when negative control (lane 3), whereas, the positive control for the PCR overexpressed in NIH3T3 cells and other cells (Lee and reaction using a KiSS-1 promoter expressing construct is seen in Welch, 1997b; Stafford et al., 2002). Considering that lane 1. A portion of the chromatin-bound promoter before co-transfection of both DRIP-130 and Sp1 resulted in antibody incubation was also used as a control for the PCR an increase of KiSS-1 expression (Figure 2b), functional reaction (lane 2). assays measuring the degree of invasive and migratory behavior of melanoma cells were used to determine DRIP-130 co-transfected A375SM cells showed similar whether re-introduction of DRIP-130 and Sp1 in highly results (data not shown), suggesting that direct interac- metastatic melanoma was sufficient to induce the tion of Sp1 occurs within this short 50 bp region, metastatic inhibition seen with KiSS-1 overexpression. specifically the 34 bases consisting entirely of over- Wound-healing assays using WM2664 cells transfected lapping Sp1 sites. with vector-only (Figure 4a), Sp1 (Figure 4c), DRIP-130 (Figure 4d) and Sp1 and DRIP-130 (Figure 4e), were DRIP-130and Sp1 complex co-precipitates with used to determine migratory ability of cells upon chromatin at GC-rich 34-bp region of KiSS-1 promoter treatment. Results demonstrated that only DRIP-130/ in melanoma Sp1 co-transfected (Figure 4e) and KiSS-1-transfected As DRIP-130 is known to be a component of the larger (Figure 4b) melanoma cells show significant migratory CRSP co-activator complex required for Sp1 transacti- inhibition 24 h after injury. DRIP-130-transfected vation, the ability of DRIP-130 to interact with the (Figure 4d) and vector-transfected cells (Figure 4a)

Oncogene Regulation of KiSS-1 expression in melanoma by Sp1 and DRIP-130 DC Mitchell et al 1743 showed a similar amount of wound healing, whereas, Sp1-transfected cells seemed to show slightly greater migratory capacity (Figure 4c). In addition, Boyden Chamber invasion assays using similarly transfected cells revealed that cell overexpressing DRIP-130 and Sp1-modulated anti-invasive behavior comparable to KiSS-1-transfected cells (Figure 4g). These functional assays suggest that re-introducing DRIP-130 and Sp1 into highly metastatic melanoma cells results in reduced invasive and migratory behavior similar to the effect produced upon KiSS-1 overexpression.

Discussion

KiSS-1 expression is commonly lost during the progres- sion of multiple forms of cancer, and the degree to which it is lost has become a relatively accurate indicator of the severity of tumor metastasis (Lee and Welch, 1997a; Kotani et al., 2001; Sanchez-Carbayo et al., 2003; Ikeguchi et al., 2004; Masui et al., 2004). Re-introduc- tion of KiSS-1 in highly metastatic cells which have progressively lost KiSS-1 expression, results in less migration and invasion and reduced metastasis (Lee and Welch, 1997b; Kotani et al., 2001; Muir et al., 2001; Ohtaki et al., 2001; Stafford et al., 2002). Considering the importance of KiSS-1 expression in gauging metastasis, an understanding of the transcriptional regulation of KiSS-1 and the mechanisms whereby it is lost during the progression of cancer is essential. Here we demonstrate that KiSS-1 transactivation is con- trolled by the Sp1 co-activator complex DRIP-130 (Mitchell et al., 2006). Specifically, loss of DRIP-130, one of 15 subunits which collectively forms the CRSP co-activator, leads to the loss of KiSS-1 transcriptional activation in metastatic melanoma cells (Taatjes and Tjian, 2004). DRIP-130 was originally identified along with the other subunits of CRSP as being essential for Sp1- mediated gene transcription (Ryu et al., 1999). In addition, other labs have found a correlation between loss of DRIP-130 and loss of KiSS-1 expression, Figure 4 Co-transfection of DRIP-130 and Sp1 induces similar anti-migratory and anti-invasive characteristics observed upon although the exact mechanism behind this correlation KiSS-1 transfection in metastatic melanoma cells. (a–f) Wound- has not been determined (Goldberg et al., 2003). The healing assays were used to determine if overexpression of Sp1 and region encoding DRIP-130, 6q16.3–q23, is commonly DRIP-130 in melanoma cells would mimic the anti-migratory lost during progression of melanoma. In addition, this properties seen in KiSS-1-transfected cells. WM2664 cells were same region of encodes activator protein singly transfected with Sp1 or DRIP-130 (c and d, respectively) and compared to the vector-transfected negative control with little (AP)-2a´ , the expression of which has previously been or no significant difference (a). Likewise, melanoma cells were shown to regulate transcription of KiSS-1 in breast co-transfected with Sp1 and DRIP-130 (e) and showed significant cancer, suggesting that loss of this region of the genome inhibition of cell migration as compared to the negative control (a). is a key milestone in the loss of KiSS-1 expression and Wound-healing assays were also done on KiSS-1 expressing cells as a positive control (b). A typical wound seen after washing heightened metastasis (Goldberg et al., 2003; Mitchell with phosphate-buffered saline before overnight incubation (f). et al., 2006) Although the GC-rich KiSS-1 promoter (g) Results from Boyden chamber migration assays demonstrate contains multiple Sp1 sites, serial truncations and that co-transfection of Sp1 and DRIP-130 leads to significant targeted deletion of the first 100 bases of the KiSS-1 migratory inhibition compared to single transfections, similar to promoter revealed that this site mediated DRIP-130/Sp1 the inhibitory effect of KiSS-1 overexpression in the cells. transcriptional regulation (Figures 1a and 3a). The ability of both a Sp1-specific antibody to supershift the discrete 34-bp region of the KiSS-1 promoter in EMSA indicated that this element may modulate Sp1

Oncogene Regulation of KiSS-1 expression in melanoma by Sp1 and DRIP-130 DC Mitchell et al 1744 transactivation through action of DRIP-130. Sp1 and tion (Figure 5b), resulting in the loss of metastatic DRIP-130 were also found capable of forming a inhibition seen in metastatic melanoma lacking KiSS-1 complex with the KiSS-1 promoter using ChIP (Figure expression. Although DRIP-130 may be simultaneously 3c and d). Although targeted mutations of the over- activating other genes, thus indirectly impacting KiSS-1 lapping Sp1 sites were attempted, owing to the high GC- expression, the necessity of the mediator complex content, we found that generation of 100-bp constructs (containing DRIP-130) for Sp1-modulated transactiva- bearing such mutations to the KiSS-1 promoter was not tion supports the direct influence of DRIP-130 levels on feasible, therefore we are unable to determine which Sp1 KiSS-1 transcription. elements, or whether all elements in the 34 bp Sp1 cluster Sp1 regulates multiple genes expressed in both normal are essential for Sp1-mediated transcriptional control. and tumorigenic tissues (Lania et al., 1997; Suske, 1999; The data presented here, as well as data from previous Black et al., 2001; Safe and Abdelrahim, 2005). Genes publications, indicates the importance of chromosome 6 involved in cell cycle progression, cell growth and (6q16.3–q23) in maintaining KiSS-1 expression (Gold- differentiation, apoptosis and capillary growth have all berg et al., 2003; Mitchell et al., 2006). Additionally, it been shown to contain GC-rich Sp1 elements (Ryuto suggests a possible mechanism for loss of KiSS-1 during et al., 1996; Finkenzeller et al., 1997; Ji et al., 1997; the progression of metastatic melanoma, by which loss Dong et al., 1999). Recent studies indicate Sp1 expres- of 6q16.3–q23 which encodes DRIP-130 leads to sion is directly correlated to severity of multiple forms of reduced KiSS-1 expression in metastatic melanoma cancer, suggesting that Sp1 expression may also be a (Figure 5). In normal cells, DRIP-130 is expressed determining factor in tumor metastatic progression (Shi properly and interacts with other proteins in the CRSP et al., 2001; Zhu et al., 2002; Wang et al., 2003; complex that helps form the larger mediator complex, Abdelrahim et al., 2004; Jiang et al., 2004; Yao et al., which regulates Sp1-mediated transcriptional regulation 2004; Liu et al., 2005; Wang and Bannon, 2005). (Figure 5a). Mutation or loss of the region encoding Another explanation for increased Sp1 in such cases, DRIP-130 results in reduced KiSS-1 expression through however, is that the normal ratio of Sp1 expression is failure of proper CRSP and mediator complex forma- altered such as in the overexpression of thrombin receptor protease-activated receptor (PAR-1). Tellez et al. (2003) explains how an irregular ratio of AP-2a´ / Sp1 in which AP-2 is lost leads to the heightened expression of PAR-1. Considering that Sp1 levels remain relatively constant in both normal and meta- static melanoma (Figure 2), a similar mechanism by which the DRIP-130/Sp1 ratio is reduced may likewise account for the loss of key genes in tumor metastasis suppression, such as KiSS-1, and result in increased tumor metastasis. Our data supports previous studies suggesting a correlation between loss of KiSS-1 expression and loss of DRIP-130 in metastatic melanoma cells and, in the course of our studies, we have localized the responsive region of the promoter to within 34-bp of KiSS-1 promoter. Considering the growing clinical importance of KiSS-1 as an effective suppressor of metastasis, our studies suggest a mechanism for its regulation in normal tissues and offer a likely mechanism for its loss during progression of cancer metastasis.

Methods and materials

Chemicals and constructs [g-32P]adenosine triphosphate (300 Ci/mmol) was ob- Figure 5 Schematic model of DRIP-130/Sp1-mediated KiSS-1 tained from PerkinElmer Life Sciences (Wellesley, MA, transcriptional regulation. In normal skin cells in which chromo- USA). Poly(dI-dC) and T4 polynucleotide kinase were some 6 is intact (a), DRIP-130 is expressed properly and interact with other protein subunits in the CRSP complex that helps form purchased from Roche Molecular Biochemicals (India- the larger mediator complex which regulates Sp1-mediated napolis, IN, USA). Antibodies for DRIP-130, Sp1, IgG transcriptional regulation of KiSS-1 gene. (b) Mutation or loss of and actin were obtained from Santa Cruz Biotechnology the genomic portion of chromosome 6q16.3–q23 encoding DRIP- (Santa Cruz, CA, USA). Luciferase reagent and lysis 130 results in loss of KiSS-1 expression through failure of proper CRSP and mediator complex formation, resulting in loss of buffer were obtained from Promega Corp. (Madison, metastatic inhibition seen in metastatic melanoma lacking KiSS-1 WI, USA). The KiSS-1 promoter was cloned as expression. described previously (Mitchell et al., 2006). Sp1 was

Oncogene Regulation of KiSS-1 expression in melanoma by Sp1 and DRIP-130 DC Mitchell et al 1745 cloned as described previously (Tellez et al., 2003). The was incubated with antibodies to IgG, DRIP-130 and DRIP-130 construct was provided by Dr Jun Qin Sp1 overnight at 41C. The region between À1 and À156 (Baylor, Houston, TX, USA). of the KiSS-1 promoter was amplified from the immunoprecipitated chromatin using the following Cell culture and transfection primers: sense, 50-TTCTCCCCAGCTCCCTGAT-CA Dulbecco’s modified Eagle’s medium (DMEM) with CATCC-30 and antisense, 50-CTGCCTCCAGT-CACA phenol red, 100 Â antibiotics and fetal bovine serum GAGC-30. The B150-bp PCR product was resolved on (FBS) were purchased from HyClone (Logan, UT, a 2.5% agarose gel and visualized under ultraviolet USA). Media was supplemented with Hepes Buffer light. (Hyclone; Logan, UT, USA) and 100 Â minimum essential medium non-essential amino acids (Gibco; Semiquantitative RT–PCR analysis Grand Island, NY, USA). T47D and MDA-435 were Total RNA was harvested using TRIzol (Invitrogen). obtained from the American Type Culture Collection First-strand complementary DNA synthesis was per- (Manassas, VA, USA). Breast cell lines were main- formed using Moloney murine leukemia virus reverse tained as described previously (Mitchell et al., 2006). transcriptase and oligo-dT (Promega) according to the Transfection of melanoma cell lines was carried out manufacturer’s protocol. Primer sequences used for according to the manufacturer’s protocol (Invitrogen, detection of KiSS-1 transcripts were 50-GCCCACCAT Carlsbad, CA, USA). In brief, DNA was added, using a GA-ACTCACTG-30 and 50-CTGC-CCCGCACCTG Lipofectamine to DNA ratio of 2:1 in each well for CG-30. Amplified products were B400 bases in length. 6 h. Empty vector was used to normalize DNA Additionally, primers for b-actin were 50-GGCTCCG concentrations. GCATGTGCAAGGC-30 and 50-AGATTTTCTCCAT GTCGTCC-30, which resulted in PCR products of Luciferase assay B200 bases. Optimal PCR cycles required for linear Upon reaching B60% confluence, reporter gene con- amplification was determined for each (b-actin required structs were transfected. Cells were harvested after 48 h, 21–23, KiSS-1 required 24–28 cycles). PCR products and luciferase activity and b-galactosidase levels were were separated on 2% agarose gels, and quantitated then measured as previously published (Mitchell et al., using Alpha Imager software (Alpha Innotech, San 2006). Leandro, CA, USA).

Western immunoblot analysis Invasion and migration assays Melanoma cell lines (2.0 Â 107) were seeded in 100-mm A375SM and WM2664 melanoma cell lines were petri dishes and incubated overnight. Lysates were transiently transfected with DRIP-130, Sp1 or vector harvested and protein concentrations determined as pre- alone. Transfection efficiency was determined visually viously published (Mitchell et al., 2006). Ten microgram upon transfection of a green fluorescent protein-tagged of each sample was then heat denatured, and loaded vector. Cell migration assays were carried out in onto either 10 or 15% sodium dodecylsulfate-polyacry- modified Boyden chambers (Banyard et al., 2000; lamide gel electrophoresis gels, as needed. Follow- Stafford et al., 2002). Filters were incubated with ing transfer to nitrocellulose (Pall Corp., Pensacola, DMEM þ bovine serum albumin after coating them in FL, USA), membranes were blocked, and primary collagen for 1 h. Filters were then put into DMEM (1:1000) and secondary (1:10 000) antibodies were without FBS and with 0.5 ng of mouse basic fibroblast added. Proteins were detected using the Super- growth factor (bFGF). Cells were seeded at approxi- Signal West Pico Chemiluminescent substrate (Pierce, mately 20 000 cells/well on top of the filter and incubated Milwaukee, WI, USA) according to the manufacturer’s 18 h. Cells were washed, fixed, stained and counted as instructions. previously published (Banyard et al., 2000; Stafford et al., 2002) Additionally, scratch assays were carried EMSA out on Sp1/DRIP-130 co-transfected A375SM and Nuclear extracts from A375SM and WM2664 cells were WM2664 cells. Cells were allowed to grow to confluency harvested as described previously (Abdelrahim et al., in collagen-coated plates, scratched and incubated in 2004). KiSS-1 promoter-derived oligonucleotides were fresh DMEM with 0.5 ng of mouse bFGF. Pictures were annealed, 50-end labeled, and EMSA was carried out as taken after 24 h using a Nikon digital camera. described previously (Mitchell et al., 2006). DNA– protein complexes were then resolved on 5% PAGE gel. Antibody–protein complexes were observed as Acknowledgements supershifted or immunodepleted complexes. This work is partially supported by a Predoctoral Traineeship Awards (W81XWH-05-1-0353 and DAMD17-03-1-0435) from ChIP assay DOD Breast Cancer Program to DM and LS, and by the NIH ChIP was performed using A375SM and WM2664 cells grants (5R01HL064792 and 1R01CA106479) to ML. We transfected with both DRIP-130 and Sp1 as described thank Dr Jun Qin at Baylor College of Medicine for the DRIP- previously (Mitchell et al., 2006). Precleared supernatant 130 construct.

Oncogene Regulation of KiSS-1 expression in melanoma by Sp1 and DRIP-130 DC Mitchell et al 1746 References

Abdelrahim M, Smith III R, Burghardt R, Safe S. (2004). Role metastasis-suppressor locus maps to 6q16.3–q23. Int J of Sp proteins in regulation of vascular endothelial growth Cancer 86: 524–528. factor expression and proliferation of pancreatic cancer Mitchell DC, Abdelrahim M, Weng J, Stafford LJ, Safe S, cells. Cancer Res 64: 6740–6749. Bar-Eli M et al. (2006). Regulation of KiSS-1 metastasis Banyard J, Anand-Apte B, Symons M, Zetter BR. (2000). suppressor gene expression in breast cancer cells by direct Motility and invasion are differentially modulated by Rho interaction of transcription factors activator protein-2alpha family GTPases. Oncogene 19: 580–591. and specificity protein-1. J Biol Chem 281: 51–58. Black AR, Black JD, Azizkhan-Clifford J. (2001). Sp1 and Muir AI, Chamberlain L, Elshourbagy NA, Michalovich D, kruppel-like factor family of transcription factors in cell Moore DJ, Calamari A et al. (2001). AXOR12, a novel growth regulation and cancer. J Cell Physiol 188: 143–160. human G protein-coupled receptor, activated by the peptide Dong L, Wang W, Wang F, Stoner M, Reed JC, Harigai M KiSS-1. J Biol Chem 276: 28969–28975. et al. (1999). Mechanisms of transcriptional activation of Ohtaki T, Shintani Y, Honda S, Matsumoto H, Hori A, bcl-2 gene expression by 17beta-estradiol in breast cancer Kanehashi K et al. (2001). Metastasis suppressor gene cells. J Biol Chem 274: 32099–32107. KiSS-1 encodes peptide ligand of a G-protein-coupled Finkenzeller G, Sparacio A, Technau A, Marme D, Siemeister G. receptor. Nature 411: 613–617. (1997). Sp1 recognition sites in the proximal promoter of the Ryu S, Tjian R. (1999). Purification of transcription cofactor human vascular endothelial growth factor gene are essential complex CRSP. Proc Natl Acad Sci USA 96: 7137–7142. for platelet-derived growth factor-induced gene expression. Ryu S, Zhou S, Ladurner AG, Tjian R. (1999). The Oncogene 15: 669–676. transcriptional cofactor complex CRSP is required for Goldberg SF, Miele ME, Hatta N, Takata M, Paquette- activity of the enhancer-binding protein Sp1. Nature 397: Straub C, Freedman LP et al. (2003). Melanoma metastasis 446–450. suppression by chromosome 6: evidence for a pathway Ryuto M, Ono M, Izumi H, Yoshida S, Weich HA, Kohno K regulated by CRSP3 and TXNIP. Cancer Res 63: 432–440. et al. (1996). Induction of vascular endothelial growth factor Ikeguchi M, Yamaguchi K, Kaibara N. (2004). Clinical by tumor necrosis factor alpha in human glioma cells. significance of the loss of KiSS-1 and orphan G-protein- Possible roles of SP-1. J Biol Chem 271: 28220–28228. coupled receptor (hOT7T175) gene expression in esophageal Safe S, Abdelrahim M. (2005). Sp transcription factor family squamous cell carcinoma. Clin Cancer Res 10: 1379–1383. and its role in cancer. Eur J Cancer 41: 2438–2448. Ji C, Casinghino S, McCarthy TL, Centrella M. (1997). Sanchez-Carbayo M, Capodieci P, Cordon-Cardo C. (2003). Multiple and essential Sp1 binding sites in the promoter for Tumor suppressor role of KiSS-1 in bladder cancer: loss of transforming growth factor-beta type I receptor. J Biol KiSS-1 expression is associated with bladder cancer Chem 272: 21260–21267. progression and clinical outcome. Am J Pathol 162: Jiang Y, Berk M, Singh LS, Tan H, Yin L, Powell CT et al. 609–617. (2005). KiSS1 suppresses metastasis in human ovarian Shi Q, Le X, Abbruzzese JL, Peng Z, Qian CN, Tang H et al. cancer via inhibition of protein kinase C alpha. Clin Exp (2001). Constitutive Sp1 activity is essential for differential Metast 22: 369–376. constitutive expression of vascular endothelial growth Jiang Y, Wang L, Gong W, Wei D, Le X, Yao J et al. (2004). factor in human pancreatic adenocarcinoma. Cancer Res A high expression level of insulin-like growth factor I 61: 4143–4154. receptor is associated with increased expression of transcrip- Shirasaki F, Takata M, Hatta N, Takehara K. (2001). Loss of tion factor Sp1 and regional lymph node metastasis of expression of the metastasis suppressor gene KiSS1 during human gastric cancer. Clin Exp Metast 21: 755–764. melanoma progression and its association with LOH of Kotani M, Detheux M, Vandenbogaerde A, Communi D, chromosome 6q16.3–q23. Cancer Res 61: 7422–7425. Vanderwinden JM, Le Poul E et al. (2001). The metastasis Stafford LJ, Xia C, Ma W, Cai Y, Liu M. (2002). suppressor gene KiSS-1 encodes kisspeptins, the natural Identification and characterization of mouse metastasis- ligands of the orphan G protein-coupled receptor GPR54. suppressor KiSS1 and its G-protein-coupled receptor. J Biol Chem 276: 34631–34636. Cancer Res 62: 5399–5404. Lania L, Majello B, De Luca P. (1997). Transcriptional Steeg PS, Ouatas T, Halverson D, Palmieri D, Salerno M. regulation by the Sp family proteins. Int J Biochem Cell Biol (2003). Metastasis suppressor genes: basic biology and 29: 1313–1323. potential clinical use. Clin Breast Cancer 4: 51–62. Lee JH, Welch DR. (1997a). Identification of highly expressed Suske G. (1999). The Sp-family of transcription factors. Gene genes in metastasis-suppressed chromosome 6/human ma- 238: 291–300. lignant melanoma hybrid cells using subtractive hybridiza- Taatjes DJ, Tjian R. (2004). Structure and function of CRSP/ tion and differential display. Int J Cancer 71: 1035–1044. Med2; a promoter-selective transcriptional coactivator Lee JH, Welch DR. (1997b). Suppression of metastasis in complex. Mol Cell 14: 675–683. human breast carcinoma MDA-MB-435 cells after transfec- Tellez C, McCarty M, Ruiz M, Bar-Eli M. (2003). Loss of tion with the metastasis suppressor gene, KiSS-1. Cancer activator protein-2alpha results in overexpression of Res 57: 2384–2387. protease-activated receptor-1 and correlates with the malig- Liu YN, Lee WW, Wang CY, Chao TH, Chen Y, Chen JH. nant phenotype of human melanoma. J Biol Chem 278: (2005). Regulatory mechanisms controlling human 46632–46642. E-cadherin gene expression. Oncogene 24: 8277–8290. Wang J, Bannon MJ. (2005). Sp1 and Sp3 activate transcrip- Masui T, Doi R, Mori T, Toyoda E, Koizumi M, Kami K tion of the human dopamine transporter gene. J Neurochem et al. (2004). Metastin and its variant forms suppress 93: 474–482. migration of pancreatic cancer cells. Biochem Biophys Res Wang L, Wei D, Huang S, Peng Z, Le X, Wu TT et al. (2003). Commun 315: 85–92. Transcription factor Sp1 expression is a significant predictor Miele ME, Jewett MD, Goldberg SF, Hyatt DL, of survival in human gastric cancer. Clin Cancer Res 9: Morelli C, Gualandi F et al. (2000). A human melanoma 6371–6380.

Oncogene Regulation of KiSS-1 expression in melanoma by Sp1 and DRIP-130 DC Mitchell et al 1747 Yao JC, Wang L, Wei D, Gong W, Hassan M, Wu TT et al. Zhu GH, Lenzi M, Schwartz EL. (2002). The Sp1 transcription (2004). Association between expression of transcription factor factor contributes to the tumor necrosis factor-induced Sp1 and increased vascular endothelial growth factor expres- expression of the angiogenic factor thymidine phospho- sion, advanced stage, and poor survival in patients with rylase in human colon carcinoma cells. Oncogene 21: resected gastric cancer. Clin Cancer Res 10(Part 1): 4109–4117. 8477–8485.

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