Regulation of Matrix Metalloproteinase Genes by E2F Transcription Factors: Rb–Raf-1 Interaction As a Novel Target for Metastatic Disease

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. 2011 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 2011 American Association for Cancer Research. and metastasis (16). We find that most MMP promoters have

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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 conducted 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 40 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). phase contrast microscopy (21). Cotransfection with 0.5 mg of pRL construct containing Renilla reniformis luciferase gene was used as normalizing Collagen degradation assays control. Luciferase assays were conducted by the Dual Lucif- Collagen degradation assays were carried out as previously erase Assay System (Promega; ref. 15). Relative luciferase described (22). First, 1 mL of type IV collagen was mixed with activity was defined as the ratio of firefly luciferase activity 7 mL of 13 mmol/L HCl, then neutralized with a buffer to Renilla luciferase activity. Error bars represent SD of 3 containing 0.2 mol/L NaP04, 16.6 mL 5 mol/L NaCl, and 80 experiments. mL 0.1 N NaOH. Seven hundred microliters of this solution was

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added to 12-well tissue culture plates to obtain a final con- (Genomatix) program. Putative E2F-binding sites were centration of 1 mg/mL of collagen. Plates were incubated at observed on the promoters of all 23 MMP genes examined 37C for 2 hours to polymerize. Thirty-five thousand CCL-210 (Supplementary Table S2). Because MMP9, MMP14, and cells were placed in a 40-mL button and were left to attach for 5 MMP15 are overexpressed in a variety of metastatic tumors hours at 37C. Complete media were added, and after 4 days, including NSCLC, these promoters were studied further. cells were trypsinized, and the remaining collagen was stained MMP9, MMP14, and MMP15 promoters had 3, 5, and 4 E2F- with Coomassie Brilliant Blue for 15 minutes, then destained binding sites, respectively, upstream of the TSS within the 2 kb (30% methanol, 10% glacial acetic acid, and 60% H20). Images regions. In addition, the MMP14 promoter had 2 E2F-binding were taken with Epson Perfection V700 Photo Scanner. sites downstream of TSS (Fig. 1A). ChIP assays were conducted on asynchronously growing Proliferation assays A549 cells to assess whether E2F1 and Rb are associated with Bromodeoxyuridine (BrdUrd) labeling kits were obtained these promoters. The location of primers used is shown in Fig. from Roche. Cells were plated in poly-D-lysine–coated chamber 1A by arrows. As shown in Fig. 1B, there was a significant slides at 5,000 cells/well and serum starved for 24 hours. Cells amount of E2F1 bound to MMP9, MMP14, and MMP15 pro- were then stimulated with serum in the presence or absence of moters, and at least 2 E2F-binding sites recruited E2F1 on each 10 mg/mL mitomycin C for 3 hours, then incubated in complete promoter. As in the case of E2F-regulated proliferative pro- media. S-phase cells were visualized by microscopy and quan- moters, Rb could also be detected on most MMP promoters. tified by counting 3 fields of 100 in quadruplicate. E2F1 was present on 3 positive control promoters, DHFR, Cdc6, and Cdc25A. There was no Rb or E2F1 present on the unrelated In vivo metastasis assay c-Fos promoter, which was the negative control. There was no Five million A549 cells stably expressing firefly luciferase DNA associated with an immunoprecipitation done with an (A549-Luc-C8; Caliper) were injected into the lateral tail vein of irrelevant antibody, further establishing the specificity of the 5-week-old female severe combined immunodeficient mice assay. This experiment suggests that the E2F sites present on (SCID)/beige mice under an Institutional Animal Care and these MMP promoters can recruit E2F1 and Rb. Use Committee–approved protocol. Mice were given Dulbec- Experiments were done to assess whether these E2F- co's Phosphate Buffered Saline (DPBS):dimethyl sulfoxide binding sites were functional. Toward this purpose, A549 (DMSO) vehicle control or RRD-251 diluted with DPBS:DMSO cells were transiently transfected with luciferase reporter once per day. For bioluminescence imaging, mice were anes- constructs driven by MMP9, MMP14, and MMP15 promo- thetized and 30 mg/kg of D-luciferin in PBS was administered ters. It was found that cotransfection of E2F1 led to a by intraperitoneal injection. Ten minutes after injection, bio- significant induction of all the 3 promoters (Fig. 1C); fur- luminescence was imaged with a charge coupled device cam- thermore, cotransfection of the large pocket region of Rb or era (Caliper). Bioluminescence images were obtained with a the full-length Rb could repress the E2F1-mediated induc- 15-cm field of view, binning (resolution) factor of 8, f/stop of 1, tion. Consistent with previous studies on proliferative E2F open filter, and an imaging time of 30 seconds to 2 minutes. target genes (10, 11), overexpression of Raf-1 could relieve Bioluminescence from relative optical intensity was defined the repression mediated by Rb. Taken together, these results manually, and data were expressed as photon flux (photons suggest that the Rb–E2F pathway might regulate MMP9, sec 1 cm 2 steradian 1) and were normalized to background MMP14, and MMP15 expression. photon flux over a mouse that was not given an injection of Gelatin zymography was used to determine whether over- luciferin. expression of E2F1 enhances MMP9 gelatinase activity. Con- sistent with the transfection data, MMP9 activity was Tissue processing and immunohistochemical staining increased 1.78-fold in A549 cells and 2.54-fold in H1650 cells Lungs were fixed in 10% neutral buffered formalin after overexpressing E2F1 (Fig. 1D and E). This suggests that the necropsy before processing into paraffin blocks. Paraffin sec- endogenous MMP9 promoter is responsive to E2F1 overex- tions (5-A thick) were rehydrated and processed using hema- pression, leading to MMP9 secretion in cell lines. toxylin and eosin (H&E) staining with standard techniques. Previous studies had shown that proliferative promoters are induced mainly by the transcriptionally active family members, Statistical analysis E2Fs 1–3. To determine whether MMP genes are also regulated Statistical analysis was conducted using one-tailed Student t exclusively by E2Fs 1–3, ChIP assays were conducted on test. Values were considered significant when less than 0.05. asynchronous A549 cells. Whereas the proliferative DHFR promoter recruited only E2Fs 1 and 3, E2Fs 1–5 were recruited Results to the promoters of both MMP9 and MMP15 (Fig. 2A); E2Fs 1–4 were recruited to the MMP14 promoter. Consistent with the MMP9, MMP14, and MMP15 promoters are responsive to ChIP assay data, transient transfection experiments on A549 E2F1 and Rb cells showed that MMP promoters are significantly induced by Microarray studies had suggested that MMP genes may be E2Fs 1–5 whereas DHFR is significantly induced by E2Fs 1–3 E2F responsive (23, 24), and to explore this possibility, we (Fig. 2B and C). These data suggest that MMPs may be a new examined the promoter region 2 kb upstream of the transcrip- class of E2F target genes, which can positively respond to tion start site (TSS) of 23 MMP genes with the MatInspector E2Fs 1–5.

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A B Input E2F1 Rb Ir Ab Input E2F1 Rb Ir Ab –1,920 (–1,904) –1,667 (–1,532) MMP9 –1,201 (–1,185) –941 (–862) –518 (–502) 37 (53) MMP9 234 (250) MMP14 MMP14

MMP15 Input E2F1 Rb Ir Ab Input E2F1 Rb Ir Ab –1,625 (–1,609) DHFR –2,000 –1,600 –1,200 –800 –400 0 bp 400 –1,800 –1,400 –1,000 –600 –200 200 –1,434 (–1,418) Cdc6 –1,036 (–1,020) Cdc25A –559 (–543) c-Fos MMP15

CD12 MMP9 MMP14 MMP15 E2F1: – + – + 10 ** **P < 0.005 * * *P < 0.05 * * *** E2F1 8 * * Actin 6 * ** * * * ** 4 ** * A549 H1650 E 2 E2F1: – + Fold change in RLA Fold 0 Luc-reporter: + + + + + + + + + + + + A549 MMP9 E2F1: – + + + + + – + + + + + 1.0 1.78 Rb-LP: – – + + – – – – + + – – Raf-1: – – – + – + – – – + – + H1650 MMP9 Rb-FL: – – – – + + – – – – + + 1.0 2.54 A549 H1650

Figure 1. A, schematic representation of MMP9, MMP14, and MMP15 promoters showing potential E2F-binding sites as diamond/circle symbols. The arrows represent the position of primers spanning E2F-binding sites tested in ChIP assays. B, ChIP assays conducted on asynchronously growing A549 cells using the indicated antibodies. Sonicated genomic DNA is used for input. The numbers indicate the position in the promoter, with respect to TSS, where a putative E2F-binding site was identiﬁed. c-Fos was used as a negative control, whereas DHFR, Cdc6, and Cdc25A were used as positive controls. Irrelevant antibody was used (Ir Ab) as a negative control for immunoprecipitation (IP). C, transient transfection experiments in A549 and H1650 cells showed that E2F1 could signiﬁcantly (, P < 0.005; P < 0.05) induce MMP9, MMP14, and MMP15 promoters, and this was repressed by Rb large pocket (Rb-LP) or full-length Rb (Rb- FL); cotransfection of Raf-1 could reverse Rb-mediated repression. Control lanes had the reporter with empty vector. D, Western blotting shows overexpression of E2F1 upon transfection compared with empty vector–transfected A549 and H1650 cells. E, A549 and H1650 cells transfected with E2F1 have increased levels of MMP9 (A549: , P ¼ 0.019; H1650: , P < 0.001) gelatinase activity as seen by Coomassie-stained zymography (inverted image).

E2F1 and E2F3 are required for MMP gene expression correlating with their binding to DHFR promoter in ChIP and collagen degradation assays (Fig. 2A); depletion of E2F2 had no effect, consistent Given that E2Fs 1–5 could induce MMP9, MMP14, and with its lack of binding in the ChIP assay; nevertheless, it could MMP15 promoters in transient transfections, attempts were induce this promoter when overexpressed. This suggests that made to assess whether E2Fs regulate the expression of their E2F1, E2F3, and E2F5 are involved in transcriptional induction endogenous promoters in NSCLC cells. Toward this purpose, of MMP9 and MMP14 genes in NSCLC cells, but they may play a A549 and H1650 cells were transfected with 100 pmol of siRNAs lesser role in regulating the endogenous MMP15 promoter. To to E2Fs 1–5 or a nontargeting control siRNA. Transfection with determine whether MMP activity could be rescued by an siRNA targeting E2F1, E2F3, or E2F5 signiﬁcantly reduced the alternate E2F family member when E2F1 is depleted, A549 expression of MMP9 and MMP14 mRNA as seen by quantita- cells were transiently transfected with siRNA-targeting E2F1, tive RT-PCR, whereas E2F2 and E2F4 had no effect (Fig. 3A). then transfected with MMP-luc constructs, with or without Because E2F5 was not detected on the MMP14 promoter in the E2F3 expression vector. E2F1 depletion led to reduced MMP9 ChIP assay of site 1,657, it is possible that E2F5 is affecting the and MMP14-luc activity, and overexpression of E2F3 could promoter through a different E2F-binding site. Surprisingly, rescue MMP9 and MMP14-luc activity. Similar results were MMP15 mRNA levels were not changed when E2Fs were obtained when E2F3 was depleted, followed by overexpression depleted in either A549 cells or H1650 cells (Fig. 3B). DHFR of E2F1. MMP15-luc activity was not affected by E2F1 or E2F3 mRNA levels were reduced when E2F1 or E2F3 were depleted, depletion (Fig. 3D).

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A B 6 A549 Input Rb E2F1 E2F2 E2F3 E2F4 E2F5 Ir Ab DHFR MMP9 5 * **P < 0.005 * *P < 0.05 MMP14 4 3 MMP15 * 2 DHFR 1 Fold change in RLA Fold C-Fos 0 Control +E2F1 +E2F2 +E2F3 +E2F4 +E2F5 C 10 A549 6 A549 6 A549 * MMP9 ** MMP14 * MMP15 * 8 5 ** 5 * 4 4 * * 6 * * ** 3 * * 3 4 * * 2 2 2 1 1 Fold change in RLA Fold 0 change in RLA Fold 0 change in RLA Fold 0 Control +E2F1 +E2F2 +E2F3 +E2F4 +E2F5 Control +E2F1 +E2F2 +E2F3 +E2F4 +E2F5 Control +E2F1 +E2F2 +E2F3 +E2F4 +E2F5

Figure 2. A, MMP9, MMP14, and MMP15 are responsive to E2Fs1–5. ChIP assays were carried out on asynchronously growing A549 cells. One binding site in each promoter was analyzed: 1,920 to 1,904 in MMP9; 1,667 to 1,532 in MMP14; and 1,625 to 1,609 in MMP15. B and C, transient transfection experiments in A549 cells showed that DHFR is significantly induced by E2Fs 1–3(, P < 0.005; P < 0.05) though no significant difference with E2F4 (P ¼ 0.18) or E2F5 (P ¼ 0.47) whereas E2Fs 1–5 could significantly induce all MMP promoters. RLA, relative luciferase activity.

Recent studies suggest that in certain tumor milieus, the cell cycle (Fig. 4A and B). There was a comparable amount of ability of fibroblasts to actively degrade extracellular collagen migration in mitomycin C–treated and nontreated cells, indi- is a climacteric step that allows cancer cells to escape the cating that the observed migration was a direct result of primary tumor site (25). Because we found that NSCLC cells motility into the empty space and independent of proliferation depleted of E2F1, E2F3, or E2F5 had less MMP9 and MMP14, we (Fig. 4A). Next, to determine whether E2F depletion affected next examined whether CCL-210 lung fibroblasts had an migration, cells were transfected with siRNA to E2F1, E2F3, a impaired ability to degrade type IV collagen, when depleted combination thereof, or a nontargeting control siRNA. Serum of E2Fs 1–5. To this end, CCL-210 cells were transfected with induced migration of cells transfected with the control, non- siRNA to E2Fs 1–5 or a nontargeting control RNA and plated on targeting siRNA into the wound, but migration was signifi- type IV collagen. After 4 days, CCL-210 cells with depleted E2Fs cantly reduced in cells transfected with E2F1 and E2F3 siRNA 1–5 had less collagen degradation as indicated by Coomassie (Fig. 4B). This suggests that E2F1 and E2F3 contribute to the staining of the residual collagen, though the depletion of E2F1 migration of cells. This agrees with studies showing that E2F1 or E2F3 had the most pronounced effect (Fig. 3C). To deter- (/) mice have abnormal epidermal repair upon injury and mine whether siRNA had any effect on proliferation of CCL-210 impaired cutaneous wound healing (27). cells, cells were counted after being trypsinized off the collagen. We next examined whether invasion was affected by deple- There was no significant difference in cell number with any tion of E2F1 or E2F3 by a Boyden chamber assay. A549 cells siRNA, suggesting that CCL-210 cells grown to confluency are were transfected with siRNA targeting E2F1, E2F3, or a com- not dependent on proliferation for collagen degradation. Tak- bination thereof. As shown in Fig. 4C and D, cells that were en together, these results suggest that depletion of E2Fs in depleted of E2F1 or E2F3 had completely lost the ability to lung cells significantly diminishes MMP gene transcription invade through collagen and Matrigel-coated Transwell filters, and hinders resultant biologic processes such as collagen whereas cells transfected with a nontargeting control siRNA degradation. showed 1.8 0.4-fold invasion in serum-stimulated cells. This suggests that E2F1 or E2F3 are required for degradation of the Depletion of E2F1 or E2F3 reduces cell migration and extracellular matrix (ECM) components through the modula- invasion tion of genes involved in their degradation. There is evidence that cell migration is accomplished in part through cleavage of adherens junctions by MMPs (26). To Rb-Raf-1 disruptor, RRD-251, represses MMP determine whether E2F-mediated modulation of MMP genes transcription and inhibits invasion and migration might affect migration of A549 cells, wound-healing assays in vitro were conducted in vitro. To ensure that changes in migration Previous work in our laboratory has shown that the Raf-1 were independent of cell proliferation, asynchronous cells were kinase interacts with Rb and phosphorylates Rb early in the cell pretreated with 10 mg/mL of mitomycin C, which arrests the cycle (10). A small-molecule Rb-Raf-1 disruptor, RRD-251,

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AB 1.6 A549 1.4 H1650 MMP9 MMP9 1.4 MMP14 MMP14 MMP15 1.2 MMP15 1.2 DHFR DHFR 1.0 1.0 * 0.8 * * 0.8 * * * * * * * 0.6 0.6 * * * * * 0.4 * 0.4

Fold change in mRNA 0.2

Fold change in mRNA 0.2 0 0 siRNA:Control E2F1 E2F2 E2F3 E2F4 E2F5 siRNA:Control E2F1 E2F2 E2F3 E2F4 E2F5

CDCCL-210

Exp1 2.5 A549 Control si * E2F1 si Exp2 2.0 E2F3 si * 1.5 Exp3 * 1.0 * * * 120 100 0.5 * 80 in RLA Fold change * * 60 * 40 0 20 +E2F1: – – – – – + – – – – – + – – – – – + 0 +E2F3: – – – – + – – – – – + – – – – – + – Cells recovered (%) Cells recovered MMP9-luc MMP14-luc MMP15-luc

siRNA:Control E2F1 E2F2 E2F3 E2F4 E2F5

Figure 3. A and B, transiently transfecting 100 pmol of E2F1, E2F3, and E2F5 siRNA reduced the expression of MMP9 and MMP14 mRNA in A549 and H1650, and there was no significant difference with E2F2 or E2F4 siRNA. DHFR mRNA levels were significantly reduced by E2F1 or E2F3 siRNA (P < 0.05). MMP15 mRNA levels were not affected significantly. C, CCL-210 lung fibroblast cells depleted of E2Fs 1–5 by siRNA show less collagen degradation compared with control siRNA. The results of 3 independent experiments are shown. The cells on top of collagen were trypsinized and counted at the termination of the experiment. Depletion of E2Fs 1–5 had no effect on growth of CCL-210 cells when plated as a confluent monolayer. Images are one representative field of CCL-210 cells atop collagen, taken at 100 total magnification with phase contrast microscopy. D, MMP9 and MMP14 luciferase activity is reduced by transiently transfecting E2F1 or E2F3 siRNA, followed by transfection of MMP-luc reporters. Cotransfection with the alternate family member (E2F1 overexpression in E2F3 si cells, E2F3 overexpression in E2F1 si cells) could rescue MMP-luc activity. Basal levels of MMP15 were not affected, though both E2F1 and E2F3 overexpression could induce MMP15-luc activity (, P < 0.05). RLA, relative luciferase activity.

inhibited Rb phosphorylation, thereby keeping Rb associated sion when E2Fs were depleted. The ability of RRD-251 to inhibit with E2F1, preventing cell proliferation and tumor growth invasion of cancer cells was next examined. A549-luc-C8 cells (12, 14). We hypothesized that RRD-251 would likely inhibit were rendered quiescent by serum starvation for 24 hours and the migration of cancer cells as well, given that depletion of then stimulated with either serum alone or serum and RRD-251 E2Fs inhibited migration. Wound-healing assays conducted on for 18 hours, and invasion was measured by a Boyden chamber MDA-MB-231, MDA-MB-435, A549, and H1650 cells showed assay. It was found that RRD-251 could signiﬁcantly abrogate that treatment with RRD-251 signiﬁcantly reduced the migra- the invasive capacity of A549-luc and MDA-MB-231 cells (Fig. tion of cells (Fig. 5A). Similarly, collagen degradation was also 5D and E). Collectively, these results suggest that RRD-251 inhibited after CCL-210 cells were treated with RRD-251 (Fig. inhibits the invasion and migration of cells, and this correlates 5B). with the repression of MMP genes. To determine whether RRD-251 could prevent E2F-mediated transcription of MMP genes, quiescent A549 cells were RRD-251 inhibits metastatic lung colonization in vivo serum stimulated in the presence or absence of 20 mmol/L Given that RRD-251 could modulate MMP levels and RRD-251. MMP mRNA levels decreased after treatment with inhibit invasion and migration in vitro, we next investigated RRD-251 (Fig. 5C), comparable with the reduction in expres- whether RRD-251 could inhibit metastasis in vivo.We

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A A549 B 0 h 24 h 0 h 24 h 70 60 * ***P < 0.001 50 **P < 0.005 *P < 0.05 40 Starved 30 20 10 * 0 +Serum BrdUrd-positive cells (%) BrdUrd-positive ×100 Untreated Mitomycin C

×100 Starved +Serum Starved +Serum 90 A549 ** Untreated Mitomycin C 80 ** 70 60 50 40 C Starved +Serum Starved +Serum 30 20 10 Scratch covered (%) covered Scratch 0 Starved +Serum Starved +Serum Untreated Mitomycin C E2F1 E2F1 si Control si + E2F3 si E2F3 si 0 h 24 h ×400 2.5 A549 ***

2.0 Starved

1.5 Control si 1.0 *** *** +Serum 0.5 *** Fold change in invasion 0

Starved +Serum Starved +Serum Starved +Serum Starved +Serum Starved Control si E2F1 si E2F3 si E2F1 + E2F3 si E2F1 si +Serum

D 90 A549 Starved 80 ***

70 E2F3 si 60 50 40 +Serum 30 ** 20 ** 10 *** Scratch covered (%) covered Scratch

0 Starved Starved +Serum Starved +Serum Starved +Serum Starved +Serum Control si E2F1 si E2F3 si E2F1 + E2F3 si E2F1 + E2F3 si +Serum ×100

Figure 4. A, A549 cells that have been treated with 10 mg/mL mitomycin C have significantly reduced BrdUrd incorporation compared with untreated A549 cells (, P < 0.05). B, serum-stimulated A549 cells treated with 10 mg/mL mitomycin C have similar migratory capacity as untreated A549 cells (, P < 0.005) 100 total magnification. C, A549 cells depleted of E2F1, E2F3, or both had significantly reduced invasive properties, as seen in a Boyden chamber assay (, P < 0.001). D, depletion of E2F1, E2F3, or E2F1 and E2F3 combined significantly hinders the ability to A549 cells to migrate in response to serum (, P < 0.005; , P < 0.001).

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Exp1 Exp2 Exp3 A MDA-MB-231 MDA-MB-435 A549 H1650 B Serum

RRD-251

Starved 100 µmol/L C ***P < 0.001 1.2 A549 Serum RRD-251 20 µmol/L **P < 0.005 1.0 *P < 0.05 +Serum 0.8 * * 0.6 * 0.4 Serum + RRD-251 50 µ mol/L 0.2 100× Fold change in mRNA 0 D MMP9 MMP14 MMP15 E 2.5 A549-luc3.0 MDA-MB-231 ** 2.0 ** 2.5 2.0 1.5 1.5 1.0 *** ** 1.0 0.5 0.5 0 0 Fold change in invasion Fold change in invasion Starved +Serum Serum + Starved +Serum Serum + 50 µmol/L 50 µmol/L RRD-251 RRD-251

Figure 5. A, MDA-MB-231, MDA-MB-435, A549, and H1650 cells treated with RRD-251 show reduced migration compared with serum. B, CCL-210 cells treated with RRD-251 show reduced degradation of type IV collagen. Images show 3 independent experiments. C, A549 cells treated with RRD-251 have signiﬁcantly reduced MMP9, MMP14, and MMP15 mRNA (, P< 0.05). D and E, A549-luc-C8 and MDA-MB-231 cells have signiﬁcantly more invasion when stimulated with serum (, P < 0.005). This effect is abrogated when treated with RRD-251 (A549-luc: , P < 0.001; MDA-MB-231: , P < 0.005).

injected A549-luc-C8 cells (5 106) into the lateral tail vein factors from Rb (8, 28). It was initially believed that the of 5-week-old female, SCID/beige mice. Mice were then predominant function of E2Fs was to activate genes required randomized into either the DMSO vehicle group or the for the progression of the cell cycle through S-phase (3, 29, 30). RRD-251 group, which received intraperitoneal injection of Later studies showed that E2F transcription factors could 50 mg/kg every day for 4 weeks. Colonization of lungs was regulate a diverse number of biologic processes including cell monitored with the Caliper-IVIS 200 system after adminis- differentiation, development, apoptosis, DNA damage repair, tration of luciferin. Mice treated with RRD-251 had signif- and more recently, angiogenesis (15, 31–34). As mentioned icantly less metastasis to the lung and surrounding tissues earlier, our laboratory had shown that the signaling kinase Raf- (Fig.6AandB).Photonflux in vehicle-treated mice was 3.9 1 directly interacts with Rb early in the cell cycle; furthermore, 0.6-fold higher than in mice treated with RRD-251. To Raf-1 could phosphorylate Rb (10). This phosphorylation of Rb confirm these observations seen in vivo,lungbiolumines- by Raf-1 was necessary for the subsequent complete inactiva- cence was examined ex vivo. Mice treated with RRD-251 had tion of Rb by CDKs. Disrupting the Rb–Raf-1 interaction using 80% less lung bioluminescence (Fig. 6C and D), indicating RRD-251 could inhibit cell proliferation, adherence indepen- less metastasis. H&E staining indicates that few tumors were dent growth, and angiogenesis and prevent the growth of lung able to seed in the lungs of mice treated with RRD-251 (Fig. cancer and melanomas in xenograft models (13). Studies 6E and F). These results suggest that RRD-251 can be used to presented here show that disrupting the Rb–Raf-1 interaction inhibit metastatic growth of tumor cells in vivo. could be a fruitful way of combating metastatic colonization of tissues by cancer cells. Discussion A considerable amount of research has been dedicated to identifying novel E2F regulated genes by gene profiling arrays It is well established that CDKs phosphorylate and inactivate and ChIP arrays (ChIP on chip; refs. 35–39). In these arrays, Rb in the G1 phase of the cell cycle, releasing E2F transcription various proteins and enzymes involved in the metastatic

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Regulation of MMP Genes by Rb and E2F

A 5.5 In vivo bioluminescence B Vehicle n = 16 * P = 0.04 d 1 Wk 1 Wk 2 Wk 3 4.5 RRD-251, 50 mg/kg, n = 16 2,000

3.5 1,500

Figure 6. A, A549-luc-C8 cells were Vehicle injected into the lateral tail vein of 2.5 1,000 Mouse 6 SCID/beige mice, and animals were 500 imaged weekly for 5 weeks. Daily in photon flux 1.5 administration of 50 mg/kg RRD-251 significantly reduced change Normalized fold Counts 0.5 Color scale lung colonization ( , P < 0.05). RRD-251 Min = 100 Mouse 9 Max = 2,000 Representative images are shown in Day 0 7 14 28 35 B. C and D, at the completion of the –0.5 ex experiment, lungs were analyzed C Ex vivo lung bioluminescence D 2,000 vivo and extent of colonization 1.2 n = 8 fi quanti ed. Mice treated with RRD- 1.0 1,500 fi Vehicle 251 had signi cantly less tumor 0.8 burden 1,000 0.6 ( , P ¼ 0.015). E and F, H&E staining * P = 0.015 fi 0.4 RRD-251 con rms the presence of numerous n = 6 500 distinct metastatic foci in the lungs of in photon flux 0.2 vehicle-treated mice, shown by 0 Counts Normalized fold change Normalized fold Vehicle RRD-251 Color scale arrows, but few in mice treated with Min = 100 RRD-251. Max = 2,000 EFVehicle RRD-251 mg/kgmg/kg

spread of tumor cells were initial hits, though validation Proangiogenic factors such as VEGF and basic fibroblast studies have been lacking. Arguably, the most crucial process growth factor are normally localized to the matrix and cannot for cancer cell invasion is the physical degradation of the ECM engage receptors until freed through MMP9 cleavage (47–49) (26), but a role for E2F transcription factors in this process had Because we have previously shown that VEGF receptors, FLT-1 not been identified. It is intriguing that, at least in lung cancer and KDR, are also E2F-regulated genes, it is likely that the role cell lines, E2Fs function as transcriptional activators of MMP9, E2F has in angiogenesis is multifaceted. MMP14, and MMP15. Many MMP gene promoters have mul- These observations raise the possibility that mutations that tiple GC boxes which can bind to Sp1 and Sp3, including initiate the oncogenic process by activating the E2F transcrip- MMP9, MMP14, MMP15, and others (40). It is well established tional regulatory pathway might also contribute to subsequent that Sp1 proteins can work coordinately with E2F transcription steps of tumor progression and metastasis. There is evidence factors to regulate gene expression (41). that the Rb–E2F pathway might affect epithelial–mesenchy- Cells use enzymes including serine-, thiol-, proteinases, mal transition as well, and this requires additional investiga- heparanases, and metalloproteinases to free them from the tion (50). Taken together, these studies link the Rb–E2F cell- primary tumor locale (16). Though the activity of MMPs and cycle regulatory pathway to advanced stages of cancer devel- other metzincin family proteins are important for metastasis, opment and metastasis. The finding that disrupting the Rb– the most prognostically valuable are the MMP family (42). A Raf-1 interaction could prevent cell proliferation, angiogenesis, relevant MMP signature is MMP2, MMP9, and MMP14, which tumor growth, and now metastatic colonization of organs have been shown to correlate with advanced-stage breast suggests that targeting the Rb–E2F pathway might be a fruitful cancer morbidity and late relapse in patients with breast avenue to combat metastatic disease. cancer (43, 44). MMP14 and MMP2 have also been detected at high levels in samples of patients with NSCLC, whereas Disclosure of Potential Conflicts of Interest MMP14 and MMP15 RNA levels have been shown to correlate with human glioma grade (45, 46). Therefore, it is a possibility No potential conflicts of interest were disclosed. that E2Fs might indirectly regulate tumor metastasis as a Acknowledgments consequence of transcriptionally activating MMPs. In addition to the crucial role for MMPs in degrading the The authors thank Dr. Jouko Lohi for the kind gift of the MMP14 construct and ECM during invasion, they also play a role in angiogenesis. to Dr. Said Sebti for helpful discussions. Assistance of the Core Facilities at

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Johnson et al.

Mofﬁtt as well as Vivarium personnel at Mofﬁtt Cancer Center is greatly The costs of publication of this article were defrayed in part by the appreciated. payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this Grant Support fact.

These studies were supported by the grant CA118120 from the National Received August 4, 2011; revised November 8, 2011; accepted November 10, Cancer Institute. 2011; published OnlineFirst November 15, 2011.

References 1. Chen HZ, Tsai SY, Leone G. Emerging roles of E2Fs in cancer: an exit 21. Dasgupta P, Rizwani W, Pillai S, Kinkade R, Kovacs M, Rastogi S, et al. from cell cycle control. Nat Rev Cancer 2009;9:785–97. Nicotine induces cell proliferation, invasion and epithelial-mesenchy- 2. Hiebert SW, Chellappan SP, Horowitz JM, Nevins JR. The interaction mal transition in a variety of human cancer cell lines. Int J Cancer of RB with E2F coincides with an inhibition of the transcriptional activity 2009;124:36–45. of E2F. Genes Dev 1992;6:177–85. 22. Netzel-Arnett S, Mitola DJ, Yamada SS, Chrysovergis K, Holmbeck K, 3. Cobrinik D. Pocket proteins and cell cycle control. Oncogene Birkedal-Hansen H, et al. Collagen dissolution by keratinocytes 2005;24:2796–809. requires cell surface plasminogen activation and matrix metallopro- 4. Kinkade R, Rizwani W, Chellappan S. E2F transcription factors in teinase activity. J Biol Chem 2002;277:45154–61. cell proliferation, apoptosis, senescence and cancer. In: Kenichi 23. Stanelle J, Stiewe T, Theseling CC, Peter M, Putzer BM. Gene expres- Yoshida, editor. Control of cellular physiology by E2F transcription sion changes in response to E2F1 activation. Nucleic Acids Res factors. Trivandrum, India: Research Signpost Press; 2007. 2002;30:1859–67. p. 69–89. 24. Ma Y, Croxton R, Moorer RL Jr, Cress WD. Identification of novel E2F1- 5. Classon M, Harlow E. The retinoblastoma tumour suppressor in regulated genes by microarray. Arch Biochem Biophys 2002;399: development and cancer. Nat Rev Cancer 2002;2:910–7. 212–24. 6. Hollander MC, Blumenthal GM, Dennis PA. PTEN loss in the continuum 25. Orimo A, Weinberg RA. Stromal fibroblasts in cancer: a novel tumor- of common cancers, rare syndromes and mouse models. Nat Rev promoting cell type. Cell Cycle 2006;5:1597–601. Cancer 2011;11:289–301. 26. Page-McCaw A, Ewald AJ, Werb Z. Matrix metalloproteinases and the 7. Sherr CJ. Principles of tumor suppression. Cell 2004;116:235–46. regulation of tissue remodelling. Nat Rev Mol Cell Biol 2007;8:221–33. 8. Attwooll C, Lazzerini Denchi E, Helin K. The E2F family: specific 27. D'Souza SJ, Vespa A, Murkherjee S, Maher A, Pajak A, Dagnino L. E2F- functions and overlapping interests. EMBO J 2004;23:4709–16. 1 is essential for normal epidermal wound repair. J Biol Chem 9. Singh S, Johnson J, Chellappan S. Small molecule regulators of Rb- 2002;277:10626–32. E2F pathway as modulators of transcription. Biochim Biophys Acta 28. Musgrove EA, Caldon CE, Barraclough J, Stone A, Sutherland RL. 2010;1799:788–94. Cyclin D as a therapeutic target in cancer. Nat Rev Cancer 2011;11: 10. Wang S, Ghosh RN, Chellappan SP. Raf-1 physically interacts with Rb 558–72. and regulates its function: a link between mitogenic signaling and cell 29. Slansky JE, Farnham PJ. Transcriptional regulation of the dihydrofo- cycle regulation. Mol Cell Biol 1998;18:7487–98. late reductase gene. Bioessays 1996;18:55–62. 11. Dasgupta P, Sun J, Wang S, Fusaro G, Betts V, Padmanabhan J, et al. 30. Lukas J, Petersen BO, Holm K, Bartek J, Helin K. Deregulated expres- Disruption of the Rb–Raf-1 interaction inhibits tumor growth and sion of E2F family members induces S-phase entry and overcomes angiogenesis. Mol Cell Biol 2004;24:9527–41. p16INK4A-mediated growth suppression. Mol Cell Biol 1996;16: 12. Kinkade R, Dasgupta P, Carie A, Pernazza D, Carless M, Pillai S, et al. A 1047–57. small molecule disruptor of Rb/Raf-1 interaction inhibits cell prolifer- 31. Croxton R, Ma Y, Song L, Haura EB, Cress WD. Direct repression of the ation, angiogenesis, and growth of human tumor xenografts in nude Mcl-1 promoter by E2F1. Oncogene 2002;21:1359–69. mice. Cancer Res 2008;68:3810–8. 32. Martinez LA, Goluszko E, Chen HZ, Leone G, Post S, Lozano G, et al. 13. Singh S, Davis R, Alamanda V, Pireddu R, Pernazza D, Sebti S, et al. E2F3 is a mediator of DNA damage-induced apoptosis. Mol Cell Biol Rb-Raf-1 interaction disruptor RRD-251 induces apoptosis in meta- 2010;30:524–36. static melanoma cells and synergizes with dacarbazine. Mol Cancer 33. Chong JL, Wenzel PL, Saenz-Robles MT, Nair V, Ferrey A, Hagan JP, Ther 2010;9:3330–41. et al. E2f1-3 switch from activators in progenitor cells to repressors in 14. Davis RK, Chellappan S. Disrupting the Rb-Raf-1 interaction: a differentiating cells. Nature 2009;462:930–4. potential therapeutic target for cancer. Drug News Perspect 34. Tsai SY, Opavsky R, Sharma N, Wu L, Naidu S, Nolan E, et al. 2008;21:331–5. Mouse development with a single E2F activator. Nature 2008;454: 15. Pillai S, Kovacs M, Chellappan S. Regulation of vascular endothelial 1137–41. growth factor receptors by Rb and E2F1: role of acetylation. Cancer 35. Lavrrar JL, Farnham PJ. The use of transient chromatin immunopre- Res 2010;70:4931–40. cipitation assays to test models for E2F1-specific transcriptional 16. Kessenbrock K, Plaks V, Werb Z. Matrix metalloproteinases: regulators activation. J Biol Chem 2004;279:46343–9. of the tumor microenvironment. Cell 2010;141:52–67. 36. Weinmann AS, Bartley SM, Zhang T, Zhang MQ, Farnham PJ. Use of 17. Lohi J, Lehti K, Valtanen H, Parks WC, Keski-Oja J. Structural analysis chromatin immunoprecipitation to clone novel E2F target promoters. and promoter characterization of the human membrane-type matrix Mol Cell Biol 2001;21:6820–32. metalloproteinase-1 (MT1-MMP) gene. Gene 2000;242:75–86. 37. Jin VX, Rabinovich A, Squazzo SL, Green R, Farnham PJ. A compu- 18. Pillai S, Dasgupta P, Chellappan SP. Chromatin immunoprecipitation tational genomics approach to identify cis-regulatory modules from assays: analyzing transcription factor binding and histone modifica- chromatin immunoprecipitation microarray data–a case study using tions in vivo. Methods Mol Biol 2009;523:323–39. E2F1. Genome Res 2006;16:1585–95. 19. Dasgupta P, Rastogi S, Pillai S, Ordonez-Ercan D, Morris M, Haura E, 38. Wells J, Graveel CR, Bartley SM, Madore SJ, Farnham PJ. et al. Nicotine induces cell proliferation by beta-arrestin-mediated The identification of E2F1-specific target genes. Proc Natl Acad Sci activation of Src and Rb-Raf-1 pathways. J Clin Invest 2006;116: U S A 2002;99:3890–5. 2208–17. 39. Wells J, Yan PS, Cechvala M, Huang T, Farnham PJ. Identification of 20. Albini A, Iwamoto Y, Kleinman HK, Martin GR, Aaronson SA, Kozlowski novel pRb binding sites using CpG microarrays suggests that E2F JM, et al. A rapid in vitro assay for quantitating the invasive potential of recruits pRb to specific genomic sites during S phase. Oncogene tumor cells. Cancer Res 1987;47:3239–45. 2003;22:1445–60.

OF10 Cancer Res; 72(2) January 15, 2012 Cancer Research

Regulation of MMP Genes by Rb and E2F

40. Clark IM, Swingler TE, Sampieri CL, Edwards DR. The regulation of 47. Manenti L, Paganoni P, Floriani I, Landoni F, Torri V, Buda A, et al. matrix metalloproteinases and their inhibitors. Int J Biochem Cell Biol Expression levels of vascular endothelial growth factor, matrix metal- 2008;40:1362–78. loproteinases 2 and 9 and tissue inhibitor of metalloproteinases 1 and 2 41. Johnson DG, Schneider-Broussard R. Role of E2F in cell cycle control in the plasma of patients with ovarian carcinoma. Eur J Cancer and cancer. Front Biosci 1998;3:d447–8. 2003;39:1948–56. 42. Roy R, Yang J, Moses MA. Matrix metalloproteinases as novel bio- 48. Belotti D, Paganoni P, Manenti L, Garofalo A, Marchini S, Taraboletti markers and potential therapeutic targets in human cancer. J Clin G, et al. Matrix metalloproteinases (MMP9 and MMP2) induce the Oncol 2009;27:5287–97. release of vascular endothelial growth factor (VEGF) by ovarian 43. Curran S, Murray GI. Matrix metalloproteinases in tumour invasion and carcinoma cells: implications for ascites formation. Cancer Res metastasis. J Pathol 1999;189:300–8. 2003;63:5224–9. 44. Curran S, Murray GI. Matrix metalloproteinases: molecular aspects of 49. Sabeh F, Ota I, Holmbeck K, Birkedal-Hansen H, Soloway P, Balbin M, their roles in tumour invasion and metastasis. Eur J Cancer 2000;36: et al. Tumor cell trafﬁc through the extracellular matrix is controlled by 1621–30. the membrane-anchored collagenase MT1-MMP. J Cell Biol 2004; 45. Bonomi P. Matrix metalloproteinases and matrix metalloproteinase 167:769–81. inhibitors in lung cancer. Semin Oncol 2002;29:78–86. 50. Arima Y, Inoue Y, Shibata T, Hayashi H, Nagano O, Saya H, et al. 46. Nuttall RK, Pennington CJ, Taplin J, Wheal A, Yong VW, Forsyth PA, Rb depletion results in deregulation of E-cadherin and induction et al. Elevated membrane-type matrix metalloproteinases in gliomas of cellular phenotypic changes that are characteristic of the revealed by proﬁling proteases and inhibitors in human cancer cells. epithelial-to-mesenchymal transition. Cancer Res 2008;68:5104– Mol Cancer Res 2003;1:333–45. 12.

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Regulation of Matrix Metalloproteinase Genes by E2F Transcription Factors: Rb−Raf-1 Interaction as a Novel Target for Metastatic Disease

Jackie L. Johnson, Smitha Pillai, Danielle Pernazza, et al.

Cancer Res Published OnlineFirst November 15, 2011.

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