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Synergistic cooperation between the AP-1 and LEF-1 transcription factors in activation of the matrilysin promoter by the src oncogene: implications in cellular invasion ¹

CHRISTINE RIVAT, NATHALIE LE FLOCH, MICHÈLE SABBAH, ISABELLE TEYROL, GÉRARD REDEUILH, ERIK BRUYNEEL^*, MARC MAREEL^*, LYNN M. MATRISIAN, HOWARD C. CRAWFORD, CHRISTIAN GESPACH and SAMIR ATTOUB²

INSERM U 482, Hôpital Saint-Antoine, 75571, Paris Cedex 12, France;
^* Laboratory of Experimental Cancerology, University Hospital, B-9000 Gent, Belgium; and
Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA

²Correspondence: INSERM U482, Hôpital Saint-Antoine, 184 rue du Faubourg Saint-Antoine, 75571, Paris Cedex 12, France; Signal Transduction and Cellular Functions in Diabetes and Digestive Cancers, Hôpital Saint-Antoine, 75571 Paris Cedex 12, France. E-mail: attoub{at}st-antoine.inserm.fr

SPECIFIC AIM

The matrix metalloprotease matrilysin (MMP-7) is overexpressed in several human solid tumors and 90% adenomas from familial adenomatous polyposis patients, suggesting its critical role during early and later stages of colon cancer invasiveness and metastasis. Oncogenic src activation is frequently associated with the emergence of premalignant adenomatous polyps and progression of metastatic colon cancers. We investigated the relationships between matrilysin promoter activity and two oncogenetic defects observed in colon cancers: activation of src and impairment of the Wnt/APC/ß-catenin pathway.

PRINCIPAL FINDINGS

1. Synergistic interactions between src and LEF-1
In transiently transfected kidney and colonic cancer cells, src induces matrilysin promoter (Mp) activation through PKC/MAPK and PI3-K-dependent signaling pathways. This promoter harbors typical response elements including AP-1, ß-catenin/TCF, and Ets/PEA3 family transcription factors, playing a critical role in cancer progression. Both PEA3 and the stable mutant of ß-catenin, N89ß-cat, did not up-regulate the Mp and are not acting synergistically with src in HCT8/S11 cells (Fig. 1 A). Axin, a negative regulator of the Wnt/APC/ß-catenin signaling pathway, had no effect on the basal Mp activity and did not affect either src or LEF-1 transactivation of the Mp (Fig. 1B , left), although cotransfection of axin with the Wnt pathway reporter TopFlash/FopFlash plasmids inhibited the ß-catenin-dependent transcription (Fig. 1B , right). Thus, the Wnt/ß-catenin/LEF signaling pathway is already activated in HCT8/S11 cells. LEF-1 alone increased Mp activity (Fig. 1A ) and induced a remarkable synergistic response with oncogenic src. Taken together, our data suggest that LEF-1 is not acting on the Mp through the canonical Wnt/APC/ß-catenin pathway.

View larger version (21K): [in this window] [in a new window]   Figure 1. Critical role of the AP-1 and LEF-1 response elements for activation of the human Mp by src through a Wnt/ß-catenin-independent signaling pathway. A) Transfection of colonic HCT8/S11 cells by the -296 bp human Mp reporter gene, alone or combined with expression vectors encoding src, PEA3, N89ß-catenin, or LEF-1. B) Effect of the Wnt inhibitor axin on the activation of Mp by src and LEF-1 (left) or the TopFlash/FopFlash luciferase reporter system (right). C) Inactivating mutations were introduced in the AP-1, Ets, and Tcf binding sites of the human Mp. Cells were cotransfected by the wild-type or mutated constructs of the -296 bp Mp, together with the control vector and src expression plasmid. Values shown are means ± SE of at least 3–5 independent experiments, each performed in triplicate.

2. AP-1 signaling and cooperative interaction between c-Jun and LEF-1
Two inactivating point mutations in the AP-1 binding site abolished src responsiveness (Fig. 1C ). In contrast, inactivating mutations within the Ets and Tcf response elements did not interfere with Mp activation by src. We decided to characterize the binding properties of the AP-1 consensus sequence using gel retardation assays and nuclear extracts prepared from src-transformed HCT8/S11 and MDCKts.src cells. DNA binding activity was remarkably enhanced after overexpression of activated src (lanes 2 and 3) (Fig. 2 A). A 50-fold excess of unlabeled oligonucleotide abolished binding of nuclear factors to the wt AP-1 probe (lanes 4) (Fig. 2A ). As expected, binding was unaffected by the addition of an excess of unlabeled mutated probe (not shown). No complex was formed in the presence of the mutated AP-1 probe (mut AP-1, lanes 1–3). Since c-Jun is a prominent component of the AP-1 transcription complexes, we show here that src up-regulates c-Jun expression (Fig. 2B ). Transcriptional activation of the Mp by src resulted in a twofold increase in the matrilysin transcripts in kidney MDCKts.src cells and colonic PCmsrc cells (Fig. 2C ).

View larger version (49K): [in this window] [in a new window]   Figure 2. Activation of the c-Jun/AP-1 binding site and its interaction with LEF-1 are required for optimal activation of the human Mp by src. A) AP-1 binding at the Mp is induced by src. EMSA was performed by incubating nuclear extracts prepared from HCT8/S11 cells or kidney MDCKts.src cells with wild-type or mutated AP-1 oligonucleotides of the Mp (wt AP-1 and mut AP-1). Lanes: 1, no nuclear extracts; 2, nuclear extracts from pSGT-transfected HCT8/S11 or MDCKts.src cells incubated at the non permissive temperature 40°C for v-src activation; 3, nuclear extracts from src-transfected HCT8/S11 or MDCKts.src cells incubated at the permissive temperature 35°C for v-src activation; 4, nuclear extracts prepared from src-transformed cells incubated with a 50-fold excess of unlabeled wild-type AP-1 probe. B) Western-blot analysis of endogenous c-Jun in whole-cell lysates from MDCKts.src cells incubated at 40°C or 35°C, human colonic adenomatous polyposis epithelial cells (PC), and their src-transformed counterparts, PCmsrc. C) Increased transcription of the endogenous matrilysin gene by src. Expression levels of the matrilysin and GAPDH transcripts from PC and PCmsrc cells by RT-PCR. D) Critical role of the proximal LEF/TCF binding sites (–109 and -194) in the AP-1/LEF-1 synergism at the Mp. HCT8/S11 cells were cotransfected with the wild-type and mutated -296 bp Mp constructs, in the presence or absence (control: C) of the v-src or LEF-1 expression plasmids. E) Molecular interaction between c-Jun and LEF-1. 35S-labeled c-Jun was prepared by translation in vitro and labeled protein probe was incubated with immobilized GST-LEF-1. The input lanes contained 10% of the labeled 35S-labeled proteins.

Using the wild-type (wt), single mutant (–109 Tcf mut, -194 Tcf mut), and double mutant matrilysin promoters –109/–194 Tcf mut), we found that the LEF-1/src synergism on Mp activity is abrogated when both Tcf sites are inactivated (Fig. 2D ). Given the proximity of the AP-1 and LEF-1 binding sites in the Mp, we demonstrated that LEF-1 interacts physically with c-Jun, using GST-LEF-1 pull-down assay (Fig. 2E ). The critical role played by the AP-1 motif in the signaling network initiated by src to control Mp activity and cellular invasion was confirmed in v-src-transformed cancer cells stably transfected by the c-jun dominant negative mutant TAM-67. Finally, we demonstrated that the new src tyrosine kinase inhibitor M475271 depleted both inactive (28 kDa) and cleavage-activated forms of matrilysin (18 kDa) in colonic HT29 cells and abrogated cellular invasion in v-src-transformed cells.

CONCLUSIONS

We identify the matrilysin gene as a transcriptional target of the src oncogene via two separate pathways, namely the PKC-p42/44 MAPK and the PI3-K cascades that converge to the AP-1 response element of the matrilysin promoter (Fig. 3 ). We demonstrate a nonconventional ß-catenin-independent cooperation between LEF-1 and src to transactivate the Mp through the AP-1 motif via a physical complex between the LEF-1 and c-Jun transcription factors. It is clear that the AP-1 response element is present in several serine protease and matrix metalloprotease genes implicated in cellular invasion: urokinase plasminogen activator, MMP-1, -3, -9, -13, and MMP-7. In agreement, src induces expression of MT-MMP1, MMP-2, and MMP-9. The transforming functions of src and other oncogenes should be considered in a complex signaling network targeting the AP-1 response elements as regulators of genes involved in tumor promotion, cancer cell proliferation, survival and invasion, and tumorigenicity. We thus provide a rationale for targeting the src/AP-1/LEF-1 signaling elements in order to control the metastatic potential of invasive cancer cells (Fig. 3) . This network could be a logical new target for the design of therapeutic strategies for the prevention and treatment of colorectal cancers and solid tumors using oligo decoys, silencing RNA, and pharmacological inhibitors such as the src inhibitor M475271, able to down-regulate this src-dependent process at the matrilysin promoter and other transforming genes controlled by AP-1/LEF-1 transcription factors.

View larger version (24K): [in this window] [in a new window]   Figure 3. Schematic diagram of the relationship between src and its downstream pathways including the PKC-p42/44 MAPK and the PI3-K cascades, converging to the AP-1/LEF-1 response elements of the matrilysin promoter implicated in cellular invasion and metastasis. The activated AP-1 site is represented by the Jun-Fos heterodimers and c-Jun interacting with the LEF-1 transcription factors at the -109 and/or -194 Tcf/LEF-1 response elements.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0132fje; doi: 10.1096/fj.03-0132fje

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