Homeotic genes of invertebrates and Hox genes of vertebrates encode a family of related transcription-regulatory proteins that play essential roles in implementing developmental patterns and conferring critical positional information (15
). Loss of trithorax
in mice incurred global loss of homeotic/Hox gene expression, resulting in profound homeotic defects (41
). Hox genes located at the 3′ end of the clusters are expressed earlier and more rostrally during development than those at the 5′ end. Accordingly, deficiency in 3′ Hox genes leads to homeotic defects in the anterior part of the body (43
mice exhibit homeotic defects in skeletons and nervous systems (26
) that are also observed in mice deficient for Hoxa3
, or Hoxa5
). This demonstrates the importance of MLL cleavage in regulating the 3′ early Hox genes, resonating with our prior in vitro data showing that knockdown of taspase-1 specifically disrupts the expression of early but not late Hoxa genes (28
). Analogous to a noncleaved MLL in mammals, trxE3Drosophila
, in which the taspase-1 cleavage site was genetically deleted, exhibited selectively reduced expression of the 3′ anterior antennapedia (ANT-C
) homeotic gene, but not the 5′ posterior bithorax (BX-C
) homeotic gene (41
). Interestingly, homeotic defects observed in Mllnc/nc
mice were less penetrant and less conspicuous than those in Tasp1–/–
mice, suggestive of potential genetic complementation from other taspase-1 substrates, such as MLL2, TFIIA, and ALF.
Our generation of Mllnc/nc
animals uncovered a previously unrecognized role of MLL in the HGF-MET signaling pathway. It has been shown that proper outgrowth of CNXII in mouse embryos relies on an intact HGF-MET pathway. HGF is expressed in branchial arches, whereas MET is expressed in subpopulations of cranial motor neurons. HGF has been shown to promote neuronal induction and axon outgrowth and to stimulate Schwann cell proliferation (49
). Genetic loss of Hgf
specifically disrupts axonal outgrowth from rhombomeres 7 and 8, therefore impairing the innervation of CNXII (32
). Our findings of specific CNXII defects in Mllnc/nc
, and Tasp1–/–
mice support the notion of specialized involvement of MLL in the HGF-MET signaling pathway. Indeed, our axon outgrowth assay using hindbrain explants of rhombomeres 7 and 8 demonstrated a role of MLL in HGF-MET–induced axonal growth. In addition to controlling CNXII outgrowth, HGF and MET also regulate the migration of skeletal myoblasts to limbs, diaphragm, and tongue (34
). When the migration of myoblasts to forelimbs was examined, we observed migration defects in Mll–/–
embryos. Although genetic defects associated with Mll–/–
embryos were less profound than those of Hgf–/–
embryos, the substantial phenotypic overlap between Mll–/–
mice and Hgf–/–
mice underscores the underlying genetic linkage between MLL and the HGF-MET signaling pathway.
Our study using hepatocellular carcinoma cell lines demonstrated that MMP1 and MMP3 functioned as key downstream effectors mediating HGF-MET–induced cell invasion, the transcriptional activation of which required MLL and ETS2, but not ETS1. As MLL and ETS2 formed a complex through the less-conserved AD2, this evolved divergence could contribute to the observed specific requirement of ETS2, but not ETS1, for HGF-induced transcription of MMP1
. Therefore, ETS1 and ETS2 are more likely functionally distinct than redundant, a notion supported by prior studies in mice deficient in individual ETS genes. Ets2–/–
mice die at E8.5 due to defects in trophoblast migration, whereas Ets1–/–
mice are viable and grossly normal, except for reduced T cell numbers (50
). Along the same line, it has been proposed that ETS2, but not ETS1, functions as the critical downstream transcription factor that signals downstream to PTEN loss in facilitating mammary epithelial tumorigenesis (51
HGF-MET induced the activity of ETS2 via deterred protein degradation and enhanced transcription. This HGF-MET–induced ETS2 accumulation ultimately led to assembly of the ETS2-MLL complex on MMP1 and MMP3 promoters. ChIP assays demonstrated minimal EBS association of ETS2 and MLL before treatment with HGF, consistent with the low level of ETS2 protein present in hepatocellular carcinoma cells prior to HGF-MET signal activation. Knockdown of ETS2 affected the targeting of MLL onto the EBSs of MMP1 and MMP3, whereas knockdown of MLL had no effects on ETS2 binding. Hence, ETS2 is primarily responsible for directing the ETS2-MLL complex to the respective promoters. Although detailed molecular mechanisms concerning stability, transcription, and DNA binding of ETS2 remain to be determined, our results uncovered an ETS2-centered molecular switch whereby HGF-MET signals the increase of ETS2 protein, which results in accumulation of the ETS2-MLL complex that targets EBSs through ETS2 and methylates H3K4 through its MLL moiety, ultimately leading to activation of MMP1 and MMP3 for cell invasion (Figure B).
The HGF-MET signaling pathway plays an essential role in diverse developmental processes, and its dysregulation contributes to invasion and metastasis phenotypes of human cancers (4
). Like the other RTKs, MET has been targeted for anticancer therapies using chemical inhibitors and antagonizing peptides and/or Abs (25
). However, human malignancies with dysregulated RTK eventually become resistant to targeted therapies due to acquired mutations in the receptor itself or in downstream signaling components. Therefore, further understanding of the molecular details downstream of the HGF-MET pathway may present additional anticancer therapeutic strategies. Here, we revealed a novel HGF-MET–enhanced ETS2-MLL complex that directly activated MMP1
transcription for the invasion of hepatocellular carcinoma cells. Since MLL mainly enhances transcription through its HMT activity, inhibitors of its HMT may offer specific measures by which to interfere with MMP1 and MMP3 induction, thereby deterring the invasiveness of cancers driven by hyperactive HGF-MET signals. Furthermore, as taspase-1–mediated cleavage of MLL was required for full HMT activity of MLL, inhibition of taspase-1 may render an alternative treatment strategy.
Besides the HGF-MET pathway, EGF, VEGF, and bFGF have been reported to function upstream of ETS1 and ETS2 transcription factors (53
). These signaling pathways promote cell invasion in a remarkably similar fashion, in which MMP expression is activated via ETS transcription factors. For example, EGF transiently induces protein levels of ETS1 and ETS2 and the ensuing upregulation of MMP1, MMP9, and uPA in SK-BR3 breast cancer cells (53
). Given the critical role of MLL in HGF-MET signaling and the sharing of some downstream signaling components among different growth factor signaling pathways, MLL family proteins may participate in other growth factor signaling pathways beyond the present study. Future investigation of the potential involvement of MLL and its family members in individual growth factor signaling pathways may shed additional light on the epigenetic control of RTK signaling beyond HGF-MET.