BLJs Are Sufficient to Suppress Epithelial Invasion
We found that four BLJ proteins, Fas2, Nrg, Dlg, and Lgl, but not proteins of other epithelial junctions, are sufficient to suppress epithelial invasion in vivo. We propose that these four proteins form a supermolecular complex at the BLJ that functions to suppress epithelial invasion (Fig. 1B). The BLJ inhibits invasion by both preventing adoption of a lateralized membrane pattern and repressing motility pathways that depend on Rac signaling.
We suggest that the similar lateralized membrane pattern of BLJ mutant cells and differentiated BCs indicates that this transformation is not adequately described as loss of polarity, but rather reflects adoption of a new pattern rich in information, essential for motility. Each membrane domain in epithelial cells acts as a targeting site for specific exocytotic and endocytotic pathways (Mellman,2000; Nelson,2003). Thus, the exclusion of apical and basal domains, and the predominance of the lateral membrane, may create a unique targeting niche essential for motility. BLJ-specific docking motifs in vesicular proteins such as SNARE (Pinheiro and Montell,2004) may be important for rapid membrane cycling in moving cells (Mellman,2000). Acquisition of the lateralized niche appears to be precisely timed in BCs, but is quasi-stochastic in BLJ mutant tumors (Fig. 3A).
Interestingly, the lateral membrane of mammalian keratinocytes also plays a role in migration events that lead to de novo epithelial sheet formation (Vasioukhin et al.,2000). The presumptive lateral surface of these cells extends filopodia, which contact adjacent cells, and facilitate adherens junction formation, an essential step preceding membrane zippering. We speculate that transformation of stationary follicle cells to invasive cells may involve a similar, modified epithelial zippering process that includes the following steps. First, BLJ mutant follicle cells decrease adhesion to each other, because as we have shown, they fail to form adherens junctions. Likewise in mammals, Dlg-1 has been shown to be required for adherens junction formation (Firestein and Rongo,2001). Second, BLJ transformed cells develop novel adhesion interactions with germ cells, because they develop basolateral membrane proteins around their circumference, and because not only proteins of the adherens junction, but also those of BLJ (with the exception of Fas2) are present on germ cell membranes (data not shown). Third, the BLJ mutant cells undergo a repetitive, recursive epithelial zippering-like process, similar to that described in keratinocytes, as they invade the germ cells, but their inability to form adherens junctions drives invasion rather then epithelial zippering. A similar mechanism may also drive BC migration.
Our data do not address whether transformation to a lateralized pattern precedes activation of motility pathways or vice versa, or if these changes occur simultaneously on the path to invasion. Independent of the sequence of these cellular transformations, the data indicate that BC invasion and BLJ tumor invasion share significant molecular and functional homology, suggesting that in addition to repressing early epithelial invasion, the BLJ also plays a dominant organizing role in controlling BC and most likely other epithelial movements (Fig. 7). We propose that in both BCs and in the early epithelium, the BLJ directly suppresses invasion by organizing the cooperative activity of distinct polarity and motility pathways (Fig. 7).
Figure 7. Homology between BC and tumor invasion. Both BCs and BLJ invasive tumor cells switch to a lateralized membrane pattern. BC migration is not only under control of BC transcription factors Slbo and Jing but also depends on Fas2 loss. Early loss of BLJ proteins is sufficient to trigger invasion in the absence of Slbo or Jing, in a quasi-stochastic pattern (Fig. 3A). Motility via either pathway depends on Rac signals. Polarity and motility pathways cooperate to suppress invasion (see text).
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BLJ proteins also suppress epithelial invasion in other contexts in flies. In eye disks that express activated RasV12, loss of dlg, lgl, or scrib causes tumor metastasis (Pagliarini and Xu,2003). Surprisingly, we found that loss of scrib in the ovarian epithelium does not cause epithelial invasion. There are several possible explanations for why Scrib does not suppress invasion in the ovary. Scrib may only suppress invasion in the context of activated Ras, or its function in suppressing invasion may depend on tissue context, the eye versus the ovary. Another possibility is that some molecules of the BLJ such as Scrib may function primarily to control polarity and suppress proliferation (Bilder,2004), while others like Dlg, Lgl, Fas2, and Nrg, additionally suppress invasion.
In contrast to BLJ proteins, loss of adherens junction DE-Cad in the RasV12 eye disk (Pagliarini and Xu,2003), or in the wild-type follicular epithelium (Niewiadomska et al.,1999), does not trigger cell invasion. This appears to be at odds with studies in mammals, where E-Cad expression is down regulated at epithelial-mesenchyme transitions, and in many human metastatic cancers. However, E-Cad loss has only been shown to trigger invasion in tumor cells, suggesting that motility may depend on the context of oncogenic signals. Thus, our work suggests that at least in the Drosophila follicular epithelium, only proteins of the BLJ, and not other junction proteins, are sufficient to suppress invasion in vivo, in the absence of large-scale genomic imbalances, or additional confounding oncogene or tumor suppressor mutations.
Relevance to Human Carcinoma Invasion
Our findings in Drosophila suggest that the BLJ may act as a conserved supermolecular complex that is crucial for suppressing invasion across phylogeny. An important finding in our model is that the spatiotemporal pattern of Fas2 predicts which follicle cells will invade (Fig. 2A,B), suggesting that tightly controlled spatiotemporal patterns of BLJ activity are crucial for suppressing motility. Thus, it is probably significant that mammalian Fas2 and Nrg orthologs, NCAM and L1, are also localized to the BLJ (Marmorstein et al.,1998; Knust,2002), along with several other IgSF members implicated in invasive cancer (Fogar et al.,1997; Roesler et al.,1997; Bazzoni,2003). Direct evidence supports NCAM's importance for suppressing invasion. NCAM is down regulated in normal migrating cells, and decreasing NCAM levels are correlated with increased tumor malignancy in human pancreatic and colorectal cancer (Fogar et al.,1997; Roesler et al.,1997; Perl et al.,1999). NCAM is also progressively lost in invasive pancreatic tumors in mice, and, like Fas2, its expression is sufficient to block invasion (Perl et al.,1999). In general, BLJ proteins are conserved in vertebrate epithelia (Knust,2002), and several BLJ proteins are lost in invasive carcinomas (Fogar et al.,1997; Roesler et al.,1997; Perl et al.,1999; Bazzoni,2003; Huang et al.,2003; Schimanski et al.,2005). hDlg-1, which directly interacts with the most commonly mutated protein in colorectal cancer, APC, is lost in murine invasive ovarian cancer (Huang et al.,2003), and loss of human ortholog of lgl contributes to colorectal cancer invasion (Schimanski et al.,2005). Greater than 90% of cancers are of epithelial origin, but usually do not pose a serious threat to life until tumor cells start to invade. The BLJ may thus be an important target for anti-cancer therapeutics because it can be manipulated from the cell surface, and is sufficient to suppress epithelial invasion. As a precedent for this idea, antibodies to L1, a human ortholog of Nrg, suppress ovarian epithelial cancer dissemination (Arlt et al.,2006). Future work will determine the degree to which the BLJ is sufficient to suppress epithelial invasion in other tissues and organisms.