Published OnlineFirst November 15, 2011; DOI: 10.1158/0008-5472.CAN-11-2647 Cancer Therapeutics, Targets, and Chemical Biology Research Regulation of Matrix Metalloproteinase Genes by E2F Transcription Factors: Rb–Raf-1 Interaction as a Novel Target for Metastatic Disease Jackie L. Johnson1,3, Smitha Pillai1, Danielle Pernazza2, Saïd M. Sebti2, Nicholas J. Lawrence2, and Srikumar P. Chellappan1 Abstract The retinoblastoma (Rb)–E2F transcriptional regulatory pathway plays a major role in cell-cycle regulation, but its role in invasion and metastasis is less well understood. We find that many genes involved in the invasion of cancer cells, such as matrix metalloproteinases (MMP), have potential E2F-binding sites in their promoters. E2F- binding sites were predicted on all 23 human MMP gene promoters, many of which harbored multiple E2F- binding sites. Studies presented here show that MMP genes such as MMP9, MMP14, and MMP15 which are overexpressed in non–small cell lung cancer, have multiple E2F-binding sites and are regulated by the Rb–E2F pathway. Chromatin immunoprecipitation assays showed the association of E2F1 with the MMP9, MMP14, and MMP15 promoters, and transient transfection experiments showed that these promoters are E2F responsive. Correspondingly, depletion of E2F family members by RNA interference techniques reduced the expression of these genes with a corresponding reduction in collagen degradation activity. Furthermore, activating Rb by inhibiting the interaction of Raf-1 with Rb by using the Rb–Raf-1 disruptor RRD-251 was sufficient to inhibit MMP transcription. This led to reduced invasion and migration of cancer cells in vitro and metastatic foci development in a tail vein lung metastasis model in mice. These results suggest that E2F transcription factors may play a role in promoting metastasis through regulation of MMP genes and that targeting the Rb–Raf-1 interaction is a promising approach for the treatment of metastatic disease. Cancer Res; 72(2); 1–11. Ó2011 AACR. Introduction is mutated in a variety of cancers, whereas mutations in signaling molecules such as K-Ras, p16INK4, and PTEN that The retinoblastoma tumor suppressor protein, Rb, together affect Rb function are prevalent in almost all cancers (4–7). with the E2F transcription factors, is the main regulator of the This indicates a major role for the Rb–E2F pathway in cell-cycle mammalian cell cycle (1). Rb physically interacts with E2F 1–3 progression and oncogenesis. Furthermore, E2Fs are known to via their transcriptional activation domain, repressing their be important for proper execution of development, differen- transcriptional activity (2). In response to mitogenic signaling, tiation, apoptosis, and DNA damage repair programs (8, 9), Rb is inactivated in the G1 phase of the cell cycle in multiple establishing a larger role for E2Fs in the biology of normal waves of phosphorylation by cyclin-dependent kinases (CDK) mammalian cells and their transformation into cancer cells. 2, 4, and 6, leading to its dissociation from E2Fs 1–3 (3). This Our earlier studies had shown that the kinase Raf-1 phys- facilitates the expression of various genes that are necessary for ically interacts with Rb early in the cell cycle, facilitating Rb DNA synthesis and cell-cycle progression, including cyclin E, phosphorylation (10, 11). Disruption of the interaction of Raf-1 dihydrofolate reductase (DHFR), and DNA polymerase a (4). with Rb using the small-molecule disruptor RRD-251 pre- Not surprisingly, oncogenic mutations target the Rb–E2F vented Rb phosphorylation, cell-cycle progression, angiogen- pathway to promote cell proliferation (5). The Rb gene itself esis, and tumor growth in mouse models (12–14). It was found that RRD-251 could inhibit the expression of E2F-regulated proliferative promoters such as Cdc25A and thymidylate Authors' Affiliations: Departments of 1Tumor Biology and 2Drug Discovery synthase (TS). Interestingly, recent studies from our laboratory and 3Cancer Biology PhD Program, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, Florida have shown that E2F1 could induce VEGF receptors, FLT-1 and KDR, indicating a role for E2F1 in tumor angiogenesis as well Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). (15). Given this background, attempts were made to assess whether E2Fs can also affect the expression of genes involved Corresponding Author: Srikumar P. Chellappan, H. Lee Moffitt Cancer Center and Research Inst., 12902 Magnolia Drive, Tampa, FL 33612. in cell invasion and cancer metastasis. Toward this purpose, we Phone: 813-745-6892; Fax: 813-745-6748; E-mail: used Genomatix MatInspector software to analyze the pro- Srikumar.Chellappan@moffitt.org moters of matrix metalloproteinase (MMP) genes, which doi: 10.1158/0008-5472.CAN-11-2647 remodel the extracellular matrix and facilitate cell invasion Ó2011 American Association for Cancer Research. and metastasis (16). We find that most MMP promoters have www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2011 American Association for Cancer Research. Published OnlineFirst November 15, 2011; DOI: 10.1158/0008-5472.CAN-11-2647 Johnson et al. multiple E2F-binding sites; data presented here show that 3 Gelatin zymography MMPs that are overexpressed in non–small cell lung cancer Media was concentrated with 7-kDa molecular weight cut-off (NSCLC), namely MMP9, MMP14, and MMP15, are in fact E2F protein concentrators at 4C (Pierce) and subjected to electro- regulated. Supporting this contention, the Rb-Raf-1 disruptor, phoresis on 8% PAGE gels containing 2 mg/mL bovine skin RRD-251, which prevents Rb phosphorylation and inhibits gelatin (Sigma). Gels were washed twice with 2.5% Triton X-100 E2F1-mediated transcription, could inhibit the transcription and then incubated for 24 hours at 37oC in Tris-HCl buffer (150 of MMP genes. In addition, RRD-251 could prevent invasion in mmol/L NaCl, 10 mmol/L CaCl2, 50 mmol/L Tris-HCl pH 7.6 vitro in vivo and decrease colonization of the lung in an tail vein and 0.05% NaN3). Gels were stained with 0.2% Coomassie metastasis model. These results suggest that the Rb–E2F Brilliant Blue and destained (30% methanol, 10% glacial acetic pathway contributes to the expression of MMP genes and that acid, and 60% H20) until gelatinolytic bands could be detected. targeting this pathway might be a potential avenue to combat Gelatinolytic signals were quantified by densitometry. metastatic disease. Chromatin immunoprecipitation assays Materials and Methods Chromatin immunoprecipitation (ChIP) assays were con- ducted on asynchronous A549 cells as previously described Cell lines and reagents (18). Immunoprecipitations were done using polyclonal anti- A549 NSCLC cells were cultured in F12K medium with 10% bodies for E2Fs 1–5 and Rb (Santa Cruz Biotechnology); a serum (Cellgro). MDA-MB-231 and MDA-MB-435 human rabbit anti-mouse secondary antibody (Pierce) was used as the breast cancer cells were cultured in Dulbecco's Modified negative control. The interaction with specific promoters was Eagle's Medium (DMEM) with 10% serum. H1650 human detected by PCR with primer sequences detailed in Supple- NSCLC cells were grown in RPMI with 10% serum. A549 cells mentary Table S1. stably expressing the firefly luciferase gene (A549-luc) were obtained from Caliper and grown in RPMI with neomycin (200 siRNA transfections and real-time PCR ng/mL). For treatment with RRD-251, cells were rendered For siRNA transfections, 100 pmol of siRNAs (Santa Cruz quiescent by serum starvation for 18 hours and then grown Biotechnology) with Oligofectamine was added to cells. For in 10% serum-containing F12K medium with RRD-251. The real-time PCR (RT-PCR), total RNA was isolated by RNeasy Rb–Raf disruptor RRD-251 was prepared as described and was miniprep kit (QIAGEN) according to the manufacturer's pro- more than 99% pure as analyzed by high-performance liquid tocol, followed by first-strand cDNA synthesis using iScript chromatography (12). cDNA synthesis kit (Bio-Rad). Data were analyzed by DDCt method, where the gene of interest was normalized to 18s rRNA Cloning of MMP promoters and, then compared with the nontargeting siRNA control sam- DNA was extracted from primary aortic endothelial cells ple. Error bars represent the SD of 3 independent experiments. with standard protocols (10). Primers spanning 2 kb of the MMP9 and MMP15 promoter were used for PCR amplification Invasion assays of the fragment with HotMaster Taq (5 Prime). Primer Boyden chamber assays were used to assess the invasive sequences were as follows: MMP9 forward, 50-TACGGTGCTT- ability of A549 and MDA-MB-231 cells as described previously GACACAGTAAATC-30; MMP9 reverse, 50-CTGACTG- (19, 20). Briefly, the upper surface of the 6.5-mm filters (Corning CAGCTGCTGTTGTGG-30; MMP15 forward, 50-GCTACT- Inc.) were coated with collagen (100 mg/filter) and Matrigel (BD TTCCTTCACTGAACAGG-30; and MMP15 reverse, 50-CGAGT- Bioscience; 50 mg/filter). Twenty thousand cells were plated in GAAGTGCGACAGTGCGGCC-30. the upper chamber with 0.1% bovine serum albumin (Sigma). The fragments were then subcloned into pCR2.1 using TA Media containing 20% FBS were placed in the lower well as cloning (Invitrogen). The plasmids were digested with KpnI chemoattractant. The cells that invaded through the filters and XhoI and ligated into pGL3 basic luciferase vector (Pro- were quantified by counting 3 fields under Â40 objective mega). The MMP14 promoter was a kind gift from Dr. Jouko magnification. Lohi at The University of Helsinski (Helsinski, Finland; ref. 17). Would-healing assays Transient transfections and luciferase assays One hundred thousand A549 cells were plated in a 6-well A549 cells were transfected with 0.5 mg of MMP reporters plate (Falcon). The cells were scratched with a sterile 200-mL along with 1 mg E2F1, 2 mg of Rb large pocket or full length, and pipette tip in 3 separate places in each well, stimulated with 2 mg Raf-1 full-length expression vector with FuGENE HD serum, and the same area was examined after 24 hours with reagent in a ratio of 4 mL FuGENE to 2 mg plasmid (Roche).