MACC1 controls Met: What a difference an Sp1 site makes

Metastasis is the most frequent cause of treatment failure in colorectal cancer. Clinical and histopathological findings are not sufficient for an early identification of subjects at high-risk for metastasis. Very recently, we identified the previously undescribed gene metastasis-associated in colon cancer 1 (MACC1). 1 Expression of MACC1 in primary, not yet metastasized human tumor specimens of subjects with colon cancer classifies patients with lowversus high-risk of tumor metastasis. MACC1 is a prognostic indicator for metastasis formation and metastasis-free survival, independent of currently used UICC/AJCC tumor staging. MACC1 induces cell migration, invasion, proliferation, colony formation, wound healing and hepatocyte growth factor (HGF)-triggered scattering of colon cancer cells in cell culture. Moreover, MACC1 causes tumor growth and metastasis in several xenograft models as demonstrated by gain-of-function and loss-offunction experiments. Detailed cellular and molecular studies indicate that MACC1 acts as master regulator of the HGF/Met signaling pathway. This pathway plays an important role for cell motility, invasiveness and metastasis.2,3 HGF acts via binding to its receptor, the tyrosine kinase Met, and thereby mediates intracellular signals, enhances migration, invasion, wound healing and survival. Deregulation of HGF/Met signaling leads to tumorigenesis and metastasis. Met is acknowledged as crucial for metastasis of colorectal cancer, and is reported as prognostic marker for early stage invasion and metastasis.4,5 We have shown that Met is a transcriptional target of MACC1. MACC1 controls the Met promoter activity and expression, thereby regulates Met-mediated signaling in a decisive manner: MACC1 transfection leads to induced Met expression, MACC1 siRNA treatment results in Met knock down and Met promoter-driven reporter expression is 10-fold increased in MACC1 transfectants. These features are lost in MACC1 mutants lacking the SH3 domain or the proline-rich motif. We demonstrated that MACC1 binds to the endogenous Met promoter and confined the binding region to a 60 bp proximal promoter fragment.1 This Met promoter fragment harbors important consensus motifs, e.g., for the transcription factors specificity protein 1 (Sp1) and AP2, that were already described as Met regulators.6,7 Sp1 binds preferentially to GC-boxes with the consensus sequences 5'-G/T-GGGCGG-G/A-G/A-C/T-3' or 5'-G/T-G/A-GGCG-G/T-G/AG/A-C/T-3'. 8 Sp1 transcriptional activity can be regulated by interaction with further proteins, such as tumor suppressors and oncogenes. Here we provide evidence that MACC1 acts via a specific consensus sequence of the Met promoter, described as an Sp1 binding site. First, we demonstrate the binding not only of MACC1, but also of the transcription factor Sp1 to the endogenous Met promoter in stably MACC1-transfected SW480 colon cancer cells by chromatin immunoprecipitation (ChIP; Fig. 1A; the wild-type Met promoter constructs were kindly provided by Yohua Liu, University of Pittsburgh, PA). However, since different Sp1 sites are described within the Met promoter fragment -223 to -68, we mutated each of these motifs by site directed mutagenesis (Fig. 1B). Reporter gene analyses (chloramphenicol acetyltransferase-enzyme linked immunosorbent assay; CAT-ELISA) revealed clearly the decisive role of the Sp1-1 site (numbering according to the occurrence of the Sp1 sites in the Met promoter beginning with the 5' Sp1; -144 to -139) for the transcriptional activation of Met (Fig. 1B). Interestingly, we observed a complete loss of Met promoter activity only if Sp1-1 was mutated. We validated this finding by electrophoretic mobility shift assays (EMSA; Fig. 1C). We used five different Met promoter oligonucleotides spanning this Met promoter fragment with overlaps. MACC1 supershifts were only observed when using oligo 2 (-170 to -111), which harbors the Sp1-1 site and the Sp1-2 site (-132 to -127). However, when using oligo 5 (-141 to -82), that harbors the Sp1-2 but not the Sp1-1 site, no supershifts have been observed (Fig. 1C). These data demonstrate that the Sp1-1 site is of essential importance for the MACC1-induced Met promoter activity. The crucial role of the Sp1 sites for establishing the constitutive expression of the Met gene was previously shown using deletion and mutation of the Sp1 sites in the Met promoter.6 Moreover, Sp1 can function to control the level of Met as demonstrated by inhibition of Sp1 binding to these sites.7 However, the mechanism of how MACC1 binds to this Sp1 site is not unveiled so far. First, MACC1 might bind directly to the transcription factor Sp1, acting then via this Sp1 binding site. It was already reported that Sp1 is able to interact with further proteins, such as Egr-1 and Smad, cooperating to regulate the transcriptional activity of the Met promoter.9,10 Furthermore, MACC1 might act via one or more mediator proteins possibly complexing MACC1 and Sp1, using this Sp1 site. Protein-protein interactions involving MACC1 are possible with respect to its domain structure. MACC1 domain mutants lacking the SH3 domain or harboring a mutated prolin-rich motif abrogated the binding of MACC1 to the Met promoter as well as the transcriptional activation of Met.1 Moreover, MACC1 might act via further transcription factors. Putative factors, that are able to bind to the Sp1-1 site in the Met promoter were found by using the PROMO software (based on the 8.3 version of TRANSFAC). 11 Beside Sp1 (-144 to -139), the transcription factors ETF (-146 to 137), E2F-1 (-142 to -135), p53 (-144 to -138) and Pax-5 (-144 to -138) were identified when allowing a maximum matrix dissimilarity rate of 15%. Interestingly, a direct interaction of Sp1 with e.g., E2F-1 and p53, but also with AP2 have been reported previously.8 Finally, it should also be taken into account, that MACC1 might bind by itself to specific DNA sequences suggesting MACC1 as a direct transcription factor. Letter to the Editor