Regulation of RON Tyrosine Kinase-mediated Invasion of Breast Cancer Cells*

RON (recepteur d'origine nantais), a tyrosine kinase receptor for macrophage-stimulating protein (MSP) was implicated in tumor progression. However, it was not investigated how this important oncogene is regulated. We show that MSP promotes invasion of MDA MB 231 and MDA MB 468 but not MCF-7 breast cancer cells. Reverse transcription-PCR and Western analysis indicated the expression of RON message and protein, respectively, in MDA MB 231 and MDA MB 468 cells but not in MCF-7 cells. RON expression correlated with Sp1 expression. Initial analysis of a 1.2-kb and 400-bp RON promoter in MDA MB 231 and MDA MB 468 cells suggested the presence of all the necessary regulatory elements within 400 bp from the transcription start site. Site-directed mutagenesis of the 400-bp RON promoter revealed that the overlapping Sp1 sites at–94 (Sp1-3/4) and Sp1 site at –113 (Sp1-5) are essential for RON gene transcription. Electrophoretic mobility shift assays and chromatin immunoprecipitation analysis indicated that Sp1 binding to these sites is required for RON promoter activity. Ectopic Sp1 expression in Sp1 null SL2 cells confirmed the involvement of these Sp1 sites in the regulation of oncogenic RON tyrosine kinase. Treatment of MDA MB 231 cells with mithramycin A, an inhibitor of Sp1 binding, or siRNA knock-down of Sp1 blocked RON gene expression and MSP-mediated invasion of MDA MB 231 cells. This is the first report demonstrating a clear link between Sp1-dependent RON tyrosine kinase expression and invasion of breast carcinoma cells.

Macrophage-stimulating protein (MSP) 2 is the only known ligand for RON (recepteur d'origine nantais). MSP is an 80 kDa heterodimer consisting of a 53 kDa ␣ chain and a 30-kDa ␤ chain linked by a disulfide bond. The ␤ chain of MSP binds to RON. MSP belongs to the plasminogen-prothrombin gene family (1,2). MSP gene knock-out in mice is not lethal, indicating that MSP is not required for embryonic development and growth (3). Besides macrophages, MSP is expressed in a variety of epithelial cells. RON is initially synthesized as a single chain precursor, 170-kDa pro-RON, which is subsequently cleaved into 40-kDa ␣ chain and 150-kDa ␤ chain. The ␣ chain is completely extracellular, whereas the ␤ chain traverses the cell membrane and contains the intracellular tyrosine kinase (1). The C-terminal of RON regulates its kinase activity (4). RON forms either homodimers or heterodimers with other receptors such as c-Met and epidermal growth factor receptor (5)(6)(7). In addition to macrophages, RON is also expressed in multiple epithelial cells both malignant and nonmalignant. Homozygous deletion of RON was embryonically lethal. However, RON heterozygous mice mature normally except for an inappropriate inflammatory response (8,9). The RON protein is regulated through c-Cbl ubiquitin ligase binding to phosphorylated RON leading to endocytosis and the subsequent degradation of RON (10).
Abnormal expression of RON was reported in various cancers of epithelial origin. However, fibroblasts do not express RON. RON is moderately expressed in normal colorectal mucosa but significantly elevated in a majority of primary human colorectal adenocarcinoma samples (11). RON protein accumulation was reported to induce autophosphorylation of RON tyrosine kinase receptor and transduces signals that regulate tumorigenic activities of colon cancer cells (12). In nonsmall cell lung cancer cell lines, RON overexpression was reported in a majority of the cell lines examined. In addition, these cell lines expressed high levels of MSP ligand (13). The combination of RON overexpression and activation by MSP leads to increased invasion and resistance to apoptosis. These tumors supported by either autocrine or paracrine effects may acquire a survival advantage because of increased activation of the RON receptor by the local secretion of MSP.
Altered RON expression was noticed in bladder and ovarian cancers. RON expression was positively associated with tumor size, stage, and grade in bladder carcinomas (14). The majority of ovarian carcinoma samples showed up-regulation in RON expression with a mix of cytoplasmic and membrane staining (5). Co-expression of MSP with RON was observed in ovarian carcinomas, providing a selective growth advantage and subsequent tumor progression. RON overexpression and not mutations is associated with head and neck squamous cell carcinomas (15). Normal breast cells and benign lesions (adenomas and papillomas) express relatively low levels of RON. However, RON is highly expressed in tumor specimens (1). Further, increased RON expression correlated to phosphorylation status and invasive activity. RON expression was an independent predictor of distant relapse in node negative breast cancer (16).
Altered RON expression in cancer cells also involved generation of RON variants through mRNA splicing, alternative initiation, and protein truncation (11,17). Of the six RON variants reported, the 55-kDa short form of RON generated through alternative initiation appears to contribute to tumor progression through modulation of E-cadherin expression (18). The RON splicing variants are generated through a complex mechanism that is critical to post-transcriptional regulation of RON expression and activation.
The promoter for RON gene was partially characterized (19). Like many tyrosine kinase receptor gene promoters, RON promoter also lacks a distinct TATA box or CCAAT sequences. However, it contains several GC boxes and consensus sequences for seven Sp1-binding sites as well as four retinoblastoma control elements. RON promoter also contains three IL-6 response and two AP-2 elements. Hence, the RON gene promoter contains several important and interesting regulatory elements. The results presented in this manuscript show that MSP stimulates the invasion of MDA MB 231 and MDA MB 468 breast cancer cells, which is correlated to the expression of MSP receptor, RON tyrosine kinase. However, it was not clearly defined how this important oncogene is regulated. It was previously suggested that multiple regulatory elements are needed for full RON promoter activity and gene expression. We now demonstrate that all other regulatory elements are dispensable except the overlapping Sp1 sites at Ϫ94 bp and another Sp1 site at Ϫ113 bp for RON promoter activity and RON gene expression in breast cancer cells. The requirement of these specific Sp1 sites for RON promoter activity was also confirmed in Sp1 null SL2 cells, where ectopic Sp1 expression stimulated the activity of the wild type RON promoter severalfold in comparison with these specific mutants. In addition, treatment of MDA MB 231 cells with mithramycin A, which inhibits Sp1 binding to target promoters or siRNA-mediated Sp1 knockdown, blocked RON gene expression and MSP-induced invasion of MDA MB 231 cells. Consequently, the results presented in this manuscript demonstrate a correlation between Sp1 and RON tyrosine kinase expression and RON kinase-associated invasion of carcinoma cells.

EXPERIMENTAL PROCEDURES
Cell Culture-MCF-7, MDA MB 231, and MDA MB 468 breast cancer cells were obtained from American Type Culture Collection. Breast cancer cells were grown in McCoy's 5A medium supplemented with 10% fetal bovine serum (Atlanta Biologicals), amino acids, antibiotics, pyruvate, and vitamins (Invitrogen). All of the cell lines were cultured in a 37°C humidified atmosphere containing 5% CO 2 . Drosophila Schneider cells (SL2) were obtained from Dr. Linda De Graffenreid. SL2 cells were grown at 28°C in Schneider's Drosophila medium (Invitrogen).
Invasion Assay-The invasive behavior of breast cancer cells was analyzed in a trans-well Boyden chamber (Costar, Bethesda, MD) using a polycarbonate filter (8-m pores) and a 0.1% gelatin matrix in the upper chamber. Briefly, 3 ϫ 10 4 cells/ well were plated in Matrigel invasion upper chambers in 0.3 ml of serum-free medium. The lower chambers contained 0.7 ml of 10% fetal bovine serum medium. The cells were treated with 1 or 2.5 ng of MSP. After 24 h of incubation at 37°C the cells on the top surface of the chamber were gently removed with cotton swabs. The migrant cells on the undersurface of the membrane were fixed in 70% methanol and stained with crystal violet. Images of migrant cells were captured by a photomicroscope (Nikon Eclipse-TE 2000-U, Japan). To assess the effect of mithramycin A, an inhibitor of Sp1 binding MDA MB 231 cells were treated with 300 nM mithramycin A for 24 h, trypsinized, and plated for Matrigel invasion assay. To determine the effect of siRNA Sp1 knock-down on MSP mediated invasion, MDA MB 231 cells were transfected with 100 nM scrambled siRNA or Sp1 siRNA. 48 h following siRNA transfection cells were trypsinized and plated for Matrigel invasion assay.
Western Blot Analysis-Equal amounts of cell lysates from MCF-7, MDA MB 231, and MDA MB 468 breast cancer cells were resolved by 7.5% SDS-PAGE and Western analysis was performed as described previously (20). Rabbit anti-human RON, Sp1, and actin polyclonal antibodies were purchased from Santa Cruz Biotechnology. To analyze the effect of mithramycin A MDA MB 231 cells were treated for 24 h with varying concentrations of mithramycin A (100, 200, and 300 nM) prior to Western analysis. To determine the effect of siRNA Sp1 knock-down on RON expression, MDA MB 231 cells were transfected with 100 nM scrambled or Sp1 siRNA, and Western analysis was performed 48 h following transfection. To verify whether similar levels of Sp1 were expressed in the ectopic Sp1transfected SL2 cells, Western analysis using Sp1 and actin antibodies was performed on the total cell lysates from control pGl3, wild type, and mutant RON promoter-luciferase construct-transfected SL2 cells.
Reverse Transcription (RT)-PCR-Total RNA from MCF-7, MDA MB 231, and MDA MB 468 breast cancer cells was reverse transcribed into cDNA. PCR analysis was then performed to determine RON expression using the cDNA as templates. Primers for actin were used as a control to determine the RON expression levels. RT-PCR analysis allows a rough estimate of the differences in RON expression in the cell lines tested. A total of 30 cycles of amplification was performed. For the studies involving mithramycin A MDA MB 231 cells were treated for 24 h with varying concentrations of mithramycin A (100, 200, and 300 nM) before RNA isolation and RT-PCR analysis. Primers for RON generate a 246-bp fragment as follows: sense primers, 5Ј-AGC CCA CGC TCA GTG TCT AT-3Ј; and antisense primers, 5Ј-GGG CAC TAG GAT CAT CTG TCA-3Ј. Primers for actin generate a 621-bp fragment as follows: sense primers, 5Ј-ACA CTG TGC CCA TCT ACG AGG-3Ј; and antisense primers, 5Ј-AGG GGC CGG ACT CGT CAT ACT-3Ј.
Construction of 1.2-kb and 400-bp Wild Type/Mutant RON Promoter-Luciferase Reporter Constructs-The 1.2-kb RON promoter-CAT reporter vector was kindly provided by Dr. Richard Breathnach. This 1.2-kb RON promoter region was subcloned into pGL3 basic luciferase reporter plasmid. The 400-bp RON promoter fragment was generated by digesting the 1.2-kb RON promoter with Kpn1 and Sac1 enzymes. The gene cleaned fragment was then amplified by PCR using primers with an integrated Kpn1 and Sac1 sites and inserted into pGL3 basic vector. The Sp1 site-specific mutation constructs of the RON promoter were generated using the QuikChange sitedirected mutagenesis kit (Stratagene, La Jolla, CA) based on the 400-bp RON promoter-luciferase reporter construct as a template. The mutations were confirmed by DNA sequencing.
Luciferase Assay-Breast cancer cells or SL2 insect cells were seeded into 6-well plates at a density of 25 ϫ 10 4 cells/well the day before transfection. The wild type or mutant RON-Luc constructs (1 g) or control null vector without the RON promoter insert (pGL3) were transiently transfected into MDA MB 231 and MDA MB 468 breast cancer cells using Lipofectamine (Invitrogen). To determine the ectopic Sp1 effects on wild type/ mutant RON promoter, SL2 cells were transiently transfected with the RON promoter-luciferase reporter constructs along with pPac empty vector or pPac-Sp1 using cellfectin (Invitrogen) as a reagent. The cells were harvested 48 h after transfection, and luciferase activity was measured (Luciferase Assay System; Promega). Control vector values were deducted from pPac-Sp1 and expressed as relative units following normalization to protein levels. To evaluate the effect of ectopic Sp1 on Ϫ400-bp RON promoter in Sp1 null MCF-7 cells, MCF-7 cells were transiently transfected with the RON promoter-luciferase reporter along with cytomegalovirus empty vector or varying concentrations of CMV-Sp1.
Electrophoretic Mobility Shift Assay (EMSA)-The wild type or mutant oligonucleotides corresponding to the specific Sp1 sites on the RON promoter were end-labeled using [␥-32 P]ATP, and EMSAs were performed as described previously (21). Wherever Sp1 antibody was used, the nuclear extracts were incubated with 2 g of Sp1 antibody (anti-rabbit; Santa Cruz Biotechnology) for 15 min on ice prior to the addition of 32 Plabeled oligonucleotide. To determine the effect of mithramycin A on Sp1 binding to the RON promoter, oligonucleotide probes were preincubated for 1 h at 4°C in the presence of 300 nM mithramycin A before adding to the nuclear extracts.
Chromatin Immunoprecipitation Assay-The chromatin immunoprecipitation assay was performed as described previously (20). RON promoter primers were used to carry out PCR on DNA isolated from chromatin immunoprecipitation using control IgG or Sp1 antibodies. The optimal reaction conditions for PCR were determined for each primer pair. The parameters included denaturation at 95°C for 1 min and annealing at 64°C for 1 min followed by elongation at 72°C for 1 min. PCR products were analyzed by 2.5% agarose/ethidium bromide gel electrophoresis. The following primers were used for PCR to generate a 293-bp fragment covering the Sp1-binding sites on the RON promoter: forward, 5Ј-CTC CAA GGG CCG GAA GAG TCG GAT GG-3Ј; and reverse, 5Ј-TTA AGC AGC GGT CCC GAC AGC CCC AA-3Ј.   RON Promoter Activity in the Invasive Breast Cancer Cells-To determine whether the elevated RON expression levels in the invasive breast cancer cells were due to increased RON transcription, we have analyzed RON promoter activities using Ϫ1.2-kb full-length and Ϫ400-bp deletion construct in MDA MB 231 and MDA MB 468 breast cancer cells. The RON promoter construct was described previously (19). We have now cloned this full-length RON promoter as well as Ϫ400-bp RON deletion fragment into pGL3 luciferase reporter plasmid (Fig. 2a). The RON promoter lacks a distinct TATA box or CCAAT sequences. However, it contains several GC boxes and consensus sequences for seven Sp1-binding sites as well as four retinoblastoma control elements. In addition, RON promoter also contains three IL-6 response elements and two AP-2 elements. Sp1 has been reported to initiate transcription from promoters that are devoid of distinct TATA box. Because six of seven Sp1 sites are located within the Ϫ400-bp RON promoter, we have analyzed the activities of Ϫ1.2-kb full-length as well as Ϫ400-bp RON promoter deletion fragment. Both the Ϫ1.2-kb and Ϫ400-bp RON promoter constructs showed similar activities in the MDA MB 231 and MDA MB 468 breast cancer cells, suggesting that all of the necessary regulatory elements are present in the

Sp1 and RON Tyrosine Kinase
Ϫ400-bp region of the RON promoter (Fig. 2b). The pGL3 control vector without RON promoter sequences was not active, thus suggesting the contribution of RON promoter elements in the regulation of RON gene expression. Because MCF-7 cells lack Sp1 and RON expression, we have analyzed whether ectopic Sp1 can stimulate RON promoter activity in MCF-7 cells (Fig. 2b). A Sp1 dose-dependent increase in RON promoter activity was observed, suggesting the requirement of Sp1 for RON gene expression.
Effect of Sp1 Mutations on RON Promoter Activities in Invasive Breast Cancer Cells-The Ϫ400-bp RON promoter contains three individual Sp1 sites (Sp1-2 at Ϫ34 bp, Sp1-5 at Ϫ113 bp, and Sp1-6 at Ϫ152 bp), two overlapping Sp1 sites (Sp1-3,4 at Ϫ92 bp) upstream of the transcription start site and one Sp1 site downstream of the transcription start site (Sp1-1 at ϩ86 bp) and an AP-2 element (Ϫ107 bp). Previous reports established the importance of the Sp1 transcription factor in the initiation of transcription from promoters that are devoid of distinct TATA box. Using site-directed mutagenesis, we have generated site-specific Sp1 mutant RON promoter constructs to identify which elements are critical for the regulation of RON promoter activity. Wild type and mutant primer sequences to generate the promoter reporter plasmids were shown in Table 1. We have analyzed the activities of the Ϫ400-bp wild type (Fig. 3, second  column) and Sp1 site-specific mutant RON promoter constructs in MDA MB 231 and MDA MB 468 cells (Fig. 3). Mutations at Sp1-2 (Ϫ34 bp; fourth column) and Sp1-6 (Ϫ152 bp; seventh column) sites did not show significant change in promoter activity. However, mutation at Sp1-5 (Ϫ113 bp; sixth column) showed a dramatic reduction in the RON promoter activity followed by mutations at the overlapping Sp1 sites, Sp1-3,4 (Ϫ92 bp; fifth column). The triple mutation at Sp1-3,4, and Sp1-5 (Ϫ92 bp and Ϫ113 bp; eighth column) completely abolished the RON promoter activity with activities comparable with that of the pGL3 control vector without the RON promoter insert (first column). In contrast, mutation at the downstream of transcription start site at Sp1-1 (ϩ86 bp; third column) increased RON promoter activity. Mutation of the AP-2 element with native Sp1-binding sites did not alter RON promoter activity (data not shown). These experiments were repeated several times, and the results are very consistent.
Sp1 Site-specific DNA Binding Activities Regulate RON Promoter Activities-Gel shift analysis (EMSA) was performed to determine whether Sp1 binding at Sp1-3,4 and Sp1-5 sites of RON promoter is required for RON promoter activity, RON gene expression, and loss of binding at these specific sites because of mutations contribute to the loss of RON promoter activity in invasive breast cancer cells (Fig. 3). The Sp1-3,4 as well as Sp1-5 wild type and mutant oligonucleotide sequences corresponding to RON promoters that were used in the gel shift analysis are shown in Table 2. These oligonucleotides were endlabeled with [ 32 P]ATP, and EMSAs were performed using nuclear extracts from MDA MB 231 breast cancer cells (Fig.  4a). Binding of some minor high mobility complexes and one major low mobility complex was detected to the Sp1-3,4 and Sp1-5 oligonucleotides (lanes 1 and 5). To identify whether the FIGURE 3. Effect of Sp1 mutations on RON promoter activity. Breast cancer cells are transiently transfected with either pGL3 control vector or Ϫ400-bp wild type or mutant RON promoter-luciferase reporter constructs, and luciferase activity was measured after 48 h following normalization to protein levels. Column 1, pGL3 control vector; column 2, Ϫ400-bp wild type RON; column 3, Sp1 mutation at Sp1-1 (ϩ86 bp); column 4, Sp1 mutation at Sp1-2 (Ϫ32 bp); column 5, overlapping Sp1 mutations at Sp1-3,4 (Ϫ94 bp); column 6, Sp1 mutation at Sp1-5 (Ϫ113 bp); column 7, Sp1 mutation at Sp1-6 (Ϫ154 bp); column 8, Sp1 triple mutations at Sp1-3,4 and Sp1-5 (Ϫ94 bp and Ϫ113 bp). The result shown represents the mean of triplicates of three individual experiments Ϯ S.D. * denotes p Ͻ 0.001 (Ϫ400 bp wild type versus Ϫ400 bp Sp1 mutants).

TABLE 1 Primer sequences used to generate site mutation constructs of the RON promoter
The consensus binding sites are shown as bold letters; the mutated sequences are shown as bold italics.

Sp1 and RON Tyrosine Kinase
FEBRUARY 29, 2008 • VOLUME 283 • NUMBER 9 major low mobility complex contained Sp1, the nuclear extracts were preincubated with 2 g of Sp1 antibody prior to the addition of 32 P-labeled oligonucleotide. The migration of major low mobility complex was shifted by the Sp1 antibody in MDA MB 231 nuclear extracts, confirming that the protein-DNA complex contained Sp1 (lanes 2 and 6). Wild type unlabeled oligonucleotides competed with 32 P-labeled oligonucleotides for binding to the protein complexes (lanes 3 and 7). Further, the low mobility complex binding was not noticed in the 32 P-labeled Sp1 sequence mutant oligonucleotide (lanes 4  and 8). These data confirm the specificity of Sp1 binding to these Sp1 sites on the RON promoter. To demonstrate that Sp1 binds to RON promoter in vivo, we have carried out chromatin immunoprecipitation analysis with control IgG or Sp1 antibodies on the chromatin fragments from MCF-7, MDA MB 231, and MDA MB 468 breast cancer cells (Fig. 4b). DNA from the immunoprecipitates was isolated, and PCR using primers covering the Sp1-binding sites was performed on the isolated DNA and nonimmunoprecipitated starting chromatin material (in put DNA). RON was detected in the in put chromatin fragments from all the three cell lines. However, accumulation of RON was detected in the Sp1 immunoprecipitates from MDA MB 231 and MDA MB 468 cells but not MCF-7, where Sp1 expression was not observed by Western analysis (Fig. 1b). RON was not detected in the control IgG immunoprecipitates, demonstrating the specificity of Sp1 binding to RON promoter. To further confirm the involvement of Sp1-3,4 and Sp1-5 sites in the regulation of RON gene expression, we have analyzed the activities of Ϫ400-bp RON promoter wild type and the specific mutant constructs in Sp1 null SL2 cells (Fig. 5a). Ectopic Sp1 expression stimulated the wild type RON promoter activities severalfold in comparison with the mutant constructs, thus confirming the requirement of these specific Sp1 sites for the regulation of oncogenic RON tyrosine kinase. Western analysis indicated that similar levels of Sp1 were expressed in all of the ectopic Sp1-transfected SL2 cells (Fig. 5b).

Mithramycin A or siRNA Sp1 Knock-down Blocks RON Gene Expression and MSP-induced Invasion of Breast Cancer Cells-
Mithramycin A was previously used to demonstrate the specificity of Sp1 binding to target promoters (22,23). To further confirm the dependence of RON gene expression on Sp1, additional experiments were performed by treating MDA MB 231 cells with mithramycin A, an inhibitor of Sp1 binding. We have treated MDA MB 231 cells for 24 h with varying concentrations of mithramycin A (100 -300 nM) and performed RT-PCR analysis on the total RNA using RON and actin primers. A dose-dependent reduction in RON transcript was observed (Fig. 6a). We have next performed EMSA analysis to determine whether the loss of Sp1 binding in the presence of mithramycin A was contributing to reduction in RON expression. Nuclear extracts from MDA MB 231 cells were incubated with radiolabeled oligonucleotides corresponding to the RON promoter. Binding of some minor high mobility complexes and one major low mobility complex was detected to Sp1-3,4, Sp1-5 oligonucleotides (Fig. 6b, lanes 1 and 4). When the nuclear extracts were preincubated with Sp1 antibody prior to the addition of radiolabeled oligonucleotides, the major low mobility complex was shifted by the Sp1 antibody, indicating that the protein-DNA complex contained Sp1 (Fig. 6b, lanes 2 and 5). Preincubation of oligonucleotide probes with 300 nM mithramycin A showed a considerable reduction in the protein-DNA complex (Fig. 6b, lanes  3 and 6). This result suggests that mithramycin A reduced the binding of Sp1 to the oligonucleotide probes that contain the Sp1 consensus elements on the RON promoter. Western analysis on the total cell lysates from control and mithramycin A-treated MDA MB 231 cells indicated a dramatic decrease in the RON protein expression (Fig. 6c). To further demonstrate the specificity of Sp1 for RON expression, we have transiently transfected MDA MB 231 cells with 100 nM scrambled siRNA control or Sp1 siRNA to inhibit endogenous Sp1 expression. 48 h following transfection, we have analyzed Sp1 and RON protein expression levels. Although scrambled siRNA did not effect Sp1 and RON expression, siRNA Sp1-mediated Sp1 knock-down blocked RON expression in the MDA MB 231 cells (Fig. 6c). To determine whether mithramycin A-mediated blockade in RON gene expression abrogates MSP-induced invasion of MDA MB 231 cells, we have carried out in vitro Matrigel assay. MDA MB 231 cells were treated for 24 h with 300 nM mithramycin A, trypsinized, and plated along with . EMSAs and chromatin immunoprecipitation. a, the wild type or mutant oligonucleotides corresponding to the specific Sp1 sites on the RON promoter were end-labeled using [␥-32 P]ATP, and EMSAs were performed as described under "Experimental Procedures." b, chromatin immunoprecipitation assay on the chromatin fragments from breast cancer cells using control IgG or Sp1 antibodies was performed as described under "Experimental Procedures."

TABLE 2 Wild type and mutant oligonucleotide sequences used in the EMSAs
untreated control cells for the Matrigel assay either in the presence or absence of 2.5 ng of MSP. MSP promoted the invasion of control MDA MB 231 cells but not mithramycin A-treated cells, where RON expression was blocked (Fig. 6d) (Fig. 6d).

DISCUSSION
Tyrosine kinase receptors regulate multiple processes involved in tumor progression and metastasis and hence making them attractive targets for molecular therapy. An understanding of the molecular alterations that facilitate tumor pro-gression and metastasis will provide insight into approaches to optimize targeted therapies. The role of RON, a tyrosine kinase receptor for MSP in human epithelial cell malignancies is currently under intensive investigation. In this report, we have delineated the role of RON in acquiring the invasive phenotype of breast cancer cells and also identified the critical regulatory elements that are necessary for oncogenic RON tyrosine kinase promoter activity and gene expression.
RON is mainly transcribed at relatively low levels in normal epithelial cells and it is not expressed in fibroblasts. However, the levels of RON expression in malignant epithelial cells were shown to increase severalfold in comparison with benign epithelium (5,(11)(12)(13)15). Elevated RON expression was strongly correlated to phosphorylation and invasive activity of tumors (12), thus suggesting that increased RON expression plays a role in the progression of carcinomas to invasive-metastatic phenotypes. Mammary-specific RON receptor overexpression induced metastatic mammary tumors that has been shown to involve ␤-catenin activation (24). Our data clearly demonstrated that MSP stimulates the invasive phenotype of MDA MB 231 and MDA MB 468 breast cancer cells (Fig. 1a). RT-PCR and Western analysis indicated the presence of RON message and protein, respectively, in these breast cancer cells (Figs. 1, c and b). The acquisition of invasive phenotype is directly correlated to the expression of oncogenic RON tyrosine kinase. Altered RON expression is also accompanied by generation of biologically active RON variants through mRNA splicing in cancer cells (1). Among the RON variants reported, the 55-kDa short form of RON generated through alternative initiation appears to contribute to tumor progression through modulation of E-cadherin expression (18).
The finding that RON is abnormally expressed and activated in epithelial cancers suggests that cellular mechanisms that control RON expression are dysfunctional in primary tumors. These studies warrant investigation into the regulation of oncogenic RON tyrosine kinase. The promoter for the RON gene has been partially characterized (19). The RON promoter lacks a distinct TATA box or CCAAT sequences. However, it is GCrich and contains consensus sequences for seven Sp1-binding sites and four retinoblastoma control elements (Fig. 2a). In addition, RON promoter also contains three IL-6 response and FIGURE 5. Ectopic Sp1 effect on RON promoter activity. a, to determine the ectopic Sp1 effects on wild type/mutant RON promoter, SL2 cells were transiently transfected with the RON promoter-luciferase reporter constructs along with pPac empty vector or pPac-Sp1 using cellfectin reagent. The cells were harvested 48 h following transfection, and luciferase activity was measured. Control vector values were deducted from pPac-Sp1 values and expressed as relative units following normalization to protein levels. pGL3, control plasmid without Ϫ400-bp RON promoter; pGL3-RON, Ϫ400-bp wild type RON promoter; RON Sp1-3,4, Ϫ400-bp RON promoter with Sp1 mutations at Sp1-3,4 (Ϫ94 bp); RON Sp1-5, Ϫ400-bp RON promoter with Sp1 mutation at Sp1-5 (Ϫ113 bp); RON Sp1-3,4,5, Ϫ400-bp RON promoter with Sp1 mutations at Sp1-3,4 and Sp1-5 (Ϫ94 bp and Ϫ113 bp). b, Western analysis using Sp1 and actin antibodies was performed to verify whether similar levels of Sp1 was expressed in all the ectopic Sp1-transfected SL2 cells.
two AP-2 elements. Consequently, RON promoter contains diverse and important regulatory elements. It was previously suggested that all of these multiple regulatory elements are required for full RON promoter activity and gene expression (19). On the contrary, the results presented in this manuscript clearly demonstrated how this important oncogene is regulated. Our initial analysis indicated that Ϫ1.2-kb full-length RON promoter is active in MDA MB 231 and MDA MB 468 breast cancer cells. Previous reports documented the importance of Sp1 transcription factor in the regulation of promoters that lack distinct TATA box. Sp1 has been reported to initiate transcription from promoters that are devoid of distinct TATA box. RON promoter also belongs to this category of genes. Because six of the seven Sp1 sites are located within the Ϫ400-bp of the transcription start site of RON gene, we have analyzed the activities of Ϫ400-bp RON promoter deletion fragment in MDA MB 231 and MDA MB 468 breast cancer cells. Both the Ϫ1.2-kb and Ϫ400-bp RON promoter elements displayed similar activities in MDA MB 231 as well as MDA MB 468 breast cancer cells, thus indicating Ϫ400-bp fragment contains all the necessary elements for optimal RON promoter activity (Fig. 2b).
The Ϫ400-bp RON promoter contains four distinct Sp1 sites and two overlapping Sp1 sites (Fig. 2a). Mutational analysis revealed that all six Sp1 sites are not vital for RON promoter activity. The Sp1-binding site at Sp1-5 followed by overlapping Sp1 sites at Sp1-3,4 are critical for RON promoter activity, and triple mutations at these sites completely abolished RON promoter activity. Surprisingly, mutation at the downstream of transcription start site (ϩ86 bp) enhanced RON promoter activity, suggesting the presence of a negative regulatory element. One possibility is that another member of the Sp gene family, Sp3, binds to this element and acts as a repressor. Sp3 recognizes the same GC element as Sp1 and has similar DNA binding affinities (25). We have previously reported that although Sp1 acts as an activator of TGF-␤ receptor expression, Sp3 acts as a repressor (21). However, our analysis indicated that Sp3 has no effect on RON promoter activity (data not shown), thus ruling out this possibility. Gel shift analysis with oligonucleotides corresponding to specific Sp1-binding sites on RON promoter using competition studies with Sp1 wild type and Sp1 mutant oligonucleotides as well as supershift approach with Sp1 antibody and chromatin immunoprecipitation analysis unequivocally demonstrated the importance of Sp1 binding to these sites for RON promoter activity. Ectopic Sp1 expression in Sp1 null SL2 cells also provided additional evidence for the requirement of these specific Sp1 sites for RON promoter activity. There is a correlation between Sp1 and RON kinase expression in the invasive MDA MB 231 and MDA MB 468 FIGURE 6. Mithramycin A blocks RON gene expression and invasion. a, total RNA from control and mithramycin A-treated MDA MB 231 cells was reverse transcribed into cDNA, and PCR analysis was performed using primers for RON and actin as described under "Experimental Procedures." b, oligonucleotides corresponding to the Sp1-binding sites on RON promoter were endlabeled using [␥-32 P[ATP, and gel shift analysis was performed using nuclear extracts from MDA MB 231 cells as described under "Experimental Procedures." c, equal amounts of cell lysates from control and mithramycin A-treated MDA MB 231 cells or control, scrambled siRNA, or Sp1 siRNA were resolved by 7.5% SDS-PAGE, and Western analysis using rabbit anti-human RON and actin polyclonal antibodies was performed. d, control or mithramycin A-treated MDA MB 231 cells or control, scrambled siRNA, or Sp1 siRNA transfected MDA MB 231 cells were plated in Matrigel invasion chamber (upper panels) and treated with 2.5 ng of MSP for 24 h. The migrant cells on the undersurface of the membrane were fixed in 70% methanol and stained with crystal violet. Images of migrant cells were captured by a photomicroscope (Nikon Eclipse-TE 2000-U, 10ϫ). cells, which are absent in MSP stimulated noninvasive MCF-7 cells. Further, studies using mithramycin A, a specific blocker of Sp1 binding to target promoters or siRNA-mediated Sp1 knock-down, confirmed the requirement of Sp1 for RON gene expression and invasion of MDA MB 231 breast carcinoma cells. In summary, the results presented in this manuscript demonstrated a direct link between RON expression and breast cancer cell invasion and delineated the regulatory elements that are critical for RON promoter activity and gene expression that contributes to metastasis of carcinoma cells.