Our previous demonstration of Src/ezrin co-operativity , and the required Y477 phosphorylation of ezrin by Src in HGF-induced scattering of epithelial cells , prompted us to examine the role of Y477 ezrin in primary tumor growth, local invasion and metastasis in an in vivo model of malignant breast cancer. Y477 is of particular interest since this residue is specific for ezrin and is not present in the two other ERM proteins, radixin or moesin, that are highly homologous to ezrin [2, 6]. In this report, we show for the first time that Y477F ezrin attenuates local invasion and distant metastasis of breast carcinoma cells transplanted into the orthotopic site of recipient mice. Moreover, Y477F ezrin promotes formation of round colonies by carcinoma cells embedded in 3D Matrigel culture, compared to formation of invasive colonies by control cells. Thus, phosphorylation of Y477 ezrin plays a key role in local invasion and metastasis from the primary tumor site.
The AC2M2 breast carcinoma cell line expresses elevated Src activity, spontaneously migrates, invades through transwell cultures, and metastasizes to the lung following engraftment into the mammary fat pad [20, 32]. An approximate 3-fold over-expression of Y477F ezrin compared to endogenous ezrin was sufficient to block spontaneous migration of AC2M2 cells in a wound healing assay, consistent with a dominant inhibitory effect of the ezrin mutant. Therefore the AC2M2 cell line model is ideally suited for studying regulation of malignant breast tumor progression by phosphorylatable Y477 ezrin.
Our observed invasive colonies of pCB6 cells with cellular extensions and protrusions in 3D Matrigel cultures is similar to the aggressive "stellate" morphology identified by Han et al. in certain human breast cancer cell lines (e.g. MDA-MB-231) with basal-like characteristics . In contrast, AC2M2 cells expressing Y477F ezrin formed predominantly round colonies with no cellular extensions or protrusions. However, cell growth and lumen filling was unaffected, indicating a partial reversion of the malignant phenotype that has been described as "cyst-like" by others [37, 43].This cyst-like phenotype is similar to the "round" morphology identified by Han et al. , and has been shown to be associated with less aggressive cancers. Gene signatures corresponding to these morphologic phenotypes were shown to accurately predict clinical outcome in independent datasets of human breast cancers .
The presence of numerous actin-rich cellular protrusions, in which ezrin is localized in pCB6 colonies, is consistent with the previously reported localization of ezrin in invasive cancers [45, 46]. In contrast, Y477F ezrin colonies showed no actin-rich protrusions, indicating a loss of invasive function; while strong actin staining in the cortical region of cells was evident. Together, these findings are consistent with previous reports that Y477F ezrin promotes increased cell-cell contacts and inhibits HGF-induced cell scattering in kidney epithelial cells [2, 6]. Interestingly, pCB6 cells expressed low E-cadherin levels and undetectable N-cadherin, and lacked robust cell-cell adhesion (, data not shown). This lack of cadherin engagement and reduced cell-cell contacts is frequently associated with more aggressive human breast cancers such as the basal-like subset .
In our in vivo mammary tumor progression model, gross pathology and histological analysis showed that the majority (> 90%) of primary control tumors engrafted into mammary fatpads of recipient mice invaded into surrounding fat tissue and underlying abdominal muscle within 21 days post engraftment. In addition, marked seeding and dissemination into visceral organs including intestine, spleen, and pancreas were frequently evident. One example of perineural invasion which occurs in about 10% of human invasive breast cancer cases , was also observed. Thus, control tumors displayed a phenotype similar to clinically advanced human breast cancers, where locally invasive primary tumors tend to anchor to the chest wall and invade into underlying muscle tissue .
In contrast to control tumors, few (25%) of the Y477F ezrin tumors showed locally invasive characteristics. The majority of Y477F ezrin tumors remained circumscribed, with little invasion into surrounding stroma and abdominal wall. Interestingly, no significant effect of Y477F ezrin on the rate of primary growth of AC2M2 tumors was observed, thus mimicking the effect of this mutant on the growth behaviour of AC2M2 cells in 3D culture. This novel finding suggests that phosphorylation at a single tyrosine residue (Y477) on ezrin plays a critical role in local invasion of tumor cells, while having no detectable effect on primary tumor outgrowth.
While Y477F ezrin was found to have a marked attenuating effect on local invasion during early phases of tumor growth, a reduction in the proportion of mice with distant metastasis was also observed, though this effect failed to reach significance. However, a significant reduction in the frequency of lung lesions, an indication of metastatic tumor load, was observed in Y477F ezrin clones compared to pCB6 control cells. These findings show that Y477F ezrin significantly reduces metastatic efficiency, perhaps due to reduced rate of release of tumor cells from the primary lesion, reduced extravasation, or decreased efficiency in establishing colonies in distant organs. The presence of residual metastases in a small proportion of mice bearing Y477F ezrin-expressing tumors could indicate alternate routes of dissemination independent of Y477 ezrin function.
The mechanism by which pY477 ezrin regulates tumor invasion in AC2M2 cells is not known. However, recent evidence shows that Src interaction via phosphorylation of Y477 ezrin regulates HGF-induced scattering and tubule formation in porcine kidney epithelial cells grown in 3D collagen gels (data not shown, and ). In this model, pY477 of ezrin interacts with Fes kinase, causing its activation and localization to cell-cell contacts, and phosphorylation by Fes of junctional proteins--a necessary step for cell scattering. We can hypothesize that in our model, the formation of round colonies observed in 3D matrigel when Y477F ezrin is expressed could be due to the lack of interaction with binding partners such as Fes. Similarly, the absence of actin-rich protrusions in cells expressing Y477F ezrin could be due to the lack of interaction between ezrin and currently unidentified partners involved in actin cytoskeleton reorganization. Moreover, pY477 of ezrin may act via pathways that connect Src to receptor tyrosine kinases such as Met, resulting in invasive properties of tumor cells . Consistent with this concept is our recent demonstration of Src/ezrin co-operativity in breast epithelial cells through increased Met activation and degradation of matrix substratum, characteristic of the invasive phenotype . Further studies are required to elucidate the role of Src/ezrin pathway and Y477 ezrin phosphorylation in the invasion process.