Ron receptor overexpression in both human and murine breast cancers is associated with a highly aggressive tumor phenotype and with a high incidence of metastasis. Despite the importance of this receptor in breast cancer, downstream effectors of Ron signaling in breast cancer have not been well-characterized. Previous studies in our laboratory have shown that mammary-specific Ron overexpression in mice leads to breast tumor formation and metastasis in vivo
(Zinser et al 2006
). We also demonstrated that Ron overexpression in these breast tumors was associated with elevated levels of tyrosine phosphorylated β-catenin. In this study, we examined the significance of Ron and β-catenin in human breast cancer. Our study provides a number of novel findings. First, we show that Ron and β-catenin are coordinately elevated in human breast cancers and that overexpression of both is associated with reduced survival and increased lymph node metastasis as compared to patients with low Ron and β-catenin levels. Second, our data demonstrate that HGFL-induced Ron activation leads to β-catenin nuclear localization and increased β-catenin reporter activity, and that tyrosine residues 654 and 670 of β-catenin are important for these processes. Third, we demonstrate a loss of β-catenin transcriptional activity and cellular proliferation in breast cancer cells in response to HGFL following a Ron knockdown and that this loss can be compensated for by activation of canonical β-catenin signaling. Moreover, while mutation of tyrosine residues 654 and 670 of β-catenin leads to a loss of HGFL mediated β-catenin transcriptional activation, activation of canonical β-catenin signaling is unaffected. Finally, we also show that targeted deletion of β-catenin in Ron expressing breast cancer cells significantly diminishes mammary tumor growth that can be compensated for by exogenous expression of wild type β-catenin but not β-catenin mutated at Y654 and Y670. These studies not only document importance of β-catenin signaling downstream of Ron but point to the critical requirement of β-catenin expression in breast tumorigenesis.
In human breast cancer, our data demonstrate that Ron receptor overexpression significantly correlates with elevated β-catenin levels. In addition to Ron, there is strong evidence implicating β-catenin signaling in breast tumorigenesis. In humans, breast tumors frequently exhibit elevated levels of β-catenin with higher expression levels correlating with decreased patient survival (Lin et al 2000
). In mice, overexpression of an activated form of β-catenin leads to the development mammary hyperplasia and adenocarcinomas (Tsukamoto et al 1988
). These studies suggest that Ron receptor overexpression and activation may contribute to β-catenin signaling in breast cancer and suggests β-catenin expression and activity may serve as a biomarker for assessing the prognosis of Ron overexpressing breast cancers. Although our data set was limited in number and follow up characteristics, our studies did find that overexpression of both Ron and β-catenin are associated with reduced survival and increased lymph node metastasis within a 30 month follow-up period compared to patients with low Ron and β-catenin.
Interestingly, overexpression of other receptor tyrosine kinases including Met, EGFR and RET have been associated with increased free β-catenin pools. Moreover, tyrosine phosphorylation of β-catenin has been shown to promote β-catenin escape from the APC/Axin/GSK3β-mediated destruction and disruption of E-cadherin association and the disassembly of adherens junctions (Castellone et al 2009
, Roura et al 1999
). Utilizing in vitro
kinase assays, our data demonstrate that Ron kinase activation leads to the tyrosine phosphorylation of β-catenin and that mutation of tyrosine residues 654 and 670 to phenylalanine significantly diminishes this effect. Our data also show that Ron receptor activation by HGFL leads to the tyrosine phosphorylation, nuclear localization and activation of β-catenin in breast cancer cell lines. Furthermore, we show that mutation of β-catenin at residues 654 and 670 from tyrosine to phenylalanine leads to a loss of HGFL-induced, Ron-dependent β-catenin nuclear localization, transcriptional activity and target gene expression. We further show that targeted deletion of β-catenin in Ron expressing breast cancer cells results in decreased cell proliferation in vitro
which can be rescued by expressing wild type β-catenin but not the 654/670 mutant of β-catenin. Interestingly, while the β-catenin knockout cells grew approximately 50% less efficiently than β-catenin expressing cell in vitro
, β-catenin expression was required for growth and metastasis of Ron expressing breast cancer cells in vivo
. While these experiments demonstrate the importance of β-catenin in breast tumorigenesis in vivo
, the data from this experiment does not specify that β-catenin deletion downstream of Ron is essential for this process but that disruption or deletion of β-catenin, as can occur through alteration of multiple signaling pathways (e.g. downstream of Wnt signaling, through various receptor tyrosine kinases, or through the disruption of cadherin functions), has an important impact in breast cancer growth and metastasis in vivo
. The studies herein are consistent with reports whereby knockdown of β-catenin expression was able to reduce RET-mediated tumor growth and invasiveness of RET overexpressing NIH 3T3 cells in subcutaneous xenograft models in nude mice (Gujral et al 2008
). In addition, mutation of β-catenin at both tyrosine residues 654 and 670 was able to block hepatocyte growth factor (HGF) induced β-catenin nuclear translocation, activation and proliferation downstream of Met receptor activation in hepatic cancer cell lines (Zeng et al 2006
). Combined, these studies show that β-catenin is a critical factor downstream of a variety of receptor tyrosine kinases for promoting tumor cell proliferation. Further, our studies combined with previous reports on the Met receptor, demonstrating that WT β-catenin but not the 654/670 mutant of β-catenin can restore cellular proliferation, suggest a novel physiological role of receptor tyrosine kinases in inducing β-catenin-dependent proliferation. Our data also support the contention that β-catenin is an important downstream effector or Ron-mediated cell proliferation in vitro
and tumor growth in vivo
In addition to β-catenin accumulation, we have also shown that HGFL-induced Ron activation results in increased levels of both cyclin D1 and c-myc. The expression of cyclin D1 and c-myc in response to HGFL was dependent on both Ron and β-catenin expression as knockdown of either Ron or β-catenin in the breast cancer cells was able to inhibit cyclin D1 and c-myc induction. In addition, expression of the β-catenin 654/670 double mutant of β-catenin was also able to blunt the induction of these genes in response to HGFL stimulation. Both c-myc and cyclin D1 have been shown to be overexpressed in different human cancers including breast cancer. While amplification of the cyclin D1 gene has been observed in about 15% of breast cancers, overexpression of cyclin D1 at the mRNA and protein levels is observed in about 50% of human breast cancers and is mostly associated with ER-positive tumors (Taneja et al 2010
). Alterations in c-myc are a common event in breast cancer, although c-myc levels alone have been difficult to correlate as a predictive or prognostic factor based on the complexity of c-myc expression and activity (Hynes and Stoelzle 2009
). Interestingly both c-myc and cyclin D1 are converging effectors of both estrogen and growth factor signaling cascades in breast cancer with the potential to mediate resistance to endocrine directed therapy. It is interesting to speculate that Ron receptor overexpression might be an important marker of breast tumorigenesis and endocrine responsiveness prompting the need for a greater understanding of Ron and Ron downstream mediators in breast cancer.
In summary, our studies have identified a novel downstream pathway of Ron receptor activation that is important for breast cancer cell proliferation, tumor growth and spread. We show that HGFL stimulation of Ron induces tyrosine phosphorylation, nuclear localization and transcriptional activation of β-catenin. This association of Ron and β-catenin is observed in both human and murine breast cancers and supports the potential of anti-Ron therapy in breast cancer due to the impact of Ron receptor signaling on tumor-established pathways.