β-Catenin is required for Ron receptor-induced mammary tumorigenesis

Abstract

Our previous studies demonstrated that selective overexpression of the Ron receptor tyrosine kinase in the murine mammary epithelium leads to mammary tumor formation. Biochemical analysis of mammary tumor lysates showed that Ron overexpression was associated with increases in β-catenin expression and tyrosine phosphorylation. β-Catenin has also been shown to be regulated through tyrosine phosphorylation by the receptor tyrosine kinases Met, Fer and Fyn. However, the molecular and physiological roles of β-catenin and β-catenin tyrosine phosphorylation downstream of Ron are not known. To investigate this association, we show that Ron and β-catenin are coordinately elevated in human breast cancers. Our data also demonstrate that activation of Ron, through ligand binding by hepatocyte growth factor-like protein (HGFL), induces the tyrosine phosphorylation of β-catenin, primarily on tyrosine residues Tyr 654 and Tyr 670. In addition, HGFL-mediated Ron activation induces both β-catenin nuclear localization and transcriptional activity, with Tyr 654 and Tyr 670 residues of β-catenin being critical for these processes. We also demonstrate that a knockdown of Ron in breast cancer cell lines leads to a loss of HGFL-induced β-catenin-dependent transcriptional activation and cell growth, which can be rescued by activation of canonical Wnt/β-catenin signaling. Moreover, we show that HGFL-dependent Ron activation mediates upregulation of the β-catenin target genes cyclin D1 and c-myc, and that expression of these target genes in breast cancer cells is decreased following inhibition of Ron and/or β-catenin. Finally, we show that genetic ablation of β-catenin in Ron-expressing breast cancer cells decreases cellular proliferation in vitro, as well as mammary tumor growth and metastasis, following orthotopic transplantation into the mammary fat pad. Together, our data suggest that β-catenin is a crucial downstream regulator of Ron receptor activation and is an important mediator of mammary tumorigenesis.

Figure 1. Ron and β-catenin expression and phosphorylation in human breast cancer

A, Serial sections from human breast cancer tissue arrays were stained for Ron and β-catenin. Representative images are shown. B, Human breast cancer cell lines were screened for Ron and β-catenin expression by Western analysis. Actin was used as a loading control. C, In vitro kinase assay were performed using WT and DM β-catenin as the substrate. Flag-tagged plasmids expressing WT and DM β-catenin were transfected into HEK293 cells and the corresponding protein immunoprecipiated with an-anti-Flag antibody. Equal amounts of WT and DM β-catenin were subsequently utilized in an in vitro kinase assay with equal amounts of Ron immunoprecipitated from R7 breast cancer cell lysates or from an isotype control immunoprecipitation. A representative kinase assay is depicted demonstrating strong phosphorylation of WT β-catenin but not DM β-catenin in combination with Ron (Top). The total amount of input for Ron, β-catenin and Flag is shown as a control (Input). The data is representative of three independent experiments with similar results.

Figure 2. Ron induces β-catenin nuclear localization and tyrosine phosphorylation

A, Ron activation induces nuclear localization of endogenous β-catenin. T47D cells were treated with HGFL (+) or vehicle (−). After 2 hours of treatment, nuclear and whole cell extracts were generated and examined by Western analysis for β-catenin levels. Expression of PARP and Tubulin were utilized as loading controls for the purity of the nuclear and whole cell extracts, respectively. B, T47D overexpressing WT and DM β-catenin were lysed and immunoblotted for Flag and β-catenin expression. Actin was used as loading control. C, Ron induces nuclear localization of exogenous WT β-catenin but not the DM form of β-catenin. T47D cells stably expressing exogenous WT and DM Flag tagged β-catenin were treated with vehicle or HGFL for 2 hours and fractioned cell extracts were examined by Western analysis as in A with the inclusion of an antibody recognizing Flag tagged exogenous β-catenin. D, Quantification of the level of exogenous WT or DM β-catenin normalized to controls (PARP or Tubulin) in the nuclear, cytoplasmic or total cell lysate of vehicle or HGFL treated cells. Note, the T47D cells transfected with DM β-catenin showed attenuated levels of β-catenin nuclear localization in response to HGFL compared to controls. All results are representative of at least 4 independent experiments.

Figure 3. Ron activation induces β-catenin-dependent activity

A, Stable Ron knockdown in T47D and R7 breast cancer cells. T47D and R7 cells were transduced with Ron shRNA (shRon) or Non-target (NT) shRNA lentiviral plasmids and cells were lysed and analyzed for Ron expression by immunoblotting. Actin was used as loading control. B, Ron activation by HGFL induced β-catenin-dependent TOP-FLASH reporter activity. T47D shNT, T47D shRon, T47D-WT β-catenin, T47D-DM β-catenin, R7 shNT and R7 shRon cells were transiently transfected with a β-catenin reporter plasmid (TOP-FLASH) and a control reporter plasmid pRL-TK for normalization of transfection efficiency. Following transfection, the cells were treated with vehicle or HGFL (100ng/ml) for 2 hours or were treated with SB216763 or vehicle for 16 hours and a dual-luciferase assay was performed. Columns represent the average fold change of three independent experiments; bars represent Standard Error (SE). *, P < 0.05 compared to the corresponding vehicle treated control group. These results are representative of three independent experiments with similar results. C, HGFL-induced cyclin D1 expression in T47D cells is inhibited following a Ron knockdown. Control T47D shNT cells and T47D shRon cells were treated with or without HGFL for 2 hours. Cell lysates were generated and examined for Ron and cyclin D1 expression. Actin was used as loading control. D, SB216763 treatment rescues the proliferation defect in Ron knockdown cells. T47D shNT, T47D shRon, R7 shNT and R7 shRon cells were treated with vehicle or SB216763 (5 μM) for 72 hours. Cell number was measured utilizing a MTT colorimetric assay. For each experiment, the value of the vehicle treated shNT control group was set at 1 and relative values for the corresponding groups were determined. Columns represent the average fold change in proliferation over the control group from three independent experiments; bars represent Standard Error (SE). *, P < 0.05.

Figure 4. β-catenin deletion in breast cancer cell lines leads to a decrease in cell proliferation in vitro

A, Ron and β-catenin expression was analyzed by Western in three independent mammary tumor cell lines (β-cateninF/F) derived from MMTV-Ron expressing mice containing a Floxed β-catenin allele. Actin expression was used as a loading control. B, The β-cateninF/F cells were infected with an adenovirus expressing Cre recombinase and GFP. GFP-positive (β-catenin−/−) and negative (β-cateninF/F) cells were isolated by FACS sorting and analyzed for β-catenin expression Western analysis. C, Quantative real-time PCR was utilized to examine β-catenin mRNA expression in β-cateninF/F and β-catenin−/− cells. Relative mRNA levels are shown normalized to an internal control. D, Deletion of β-catenin leads to decreased proliferation. β-cateninF/F and β-catenin−/− cells were incubated with BrdU for 4 hours and immunocytostaining for BrdU incorporation was performed. The percent of cells straining positive for BrdU was quantitated from three independent high power fields and the data tabulated is a result from three independent experiments. *, P < 0.05. E, β-cateninF/F and β-catenin−/− cells were plated in triplicate in 24 well plates. Cell number was measured at 0, 24, 48, 72 and 96 hours by crystal violet staining assays. The graph is representative of the three independent experiments. *, P < 0.05 compared to the corresponding β-catenin−/− cells.

Figure 5. Transfection of WT β-catenin but not DM β-catenin partly rescues cell growth of β-catenin−/− cells

A, β-catenin−/− cells were transiently transfected with Flag-tagged empty vector (EV), WT, or DM β-catenin. After 48 hours, cell lysates were generated and examined for Flag expression by Western analysis. Actin expression is provided as a loading control. B, β-catenin−/− cells were transfected with Flag-tagged empty vector (EV), WT, or DM β-catenin. After 48 hours, the cell number was quantiated utilizing an MTT assay. The experiment was performed three times in triplet and the data shown represents the mean of all experiments ± SE. *, P <0.05.

Figure 6. Deletion of β-catenin prohibits mammary tumor formation and metastasis following implantation in the mammary fat pad of nude mice

A, β-cateninF/F and β-catenin−/− cells were orthotopically transplanted into the mammary fat pads of nude mice. Mammary tumor growth was measured biweekly for up to 6 weeks and tumor volume was plotted over time. B, Mammary tumor weight of mice transplanted with β-catenin−/− and β-cateninF/F cells at 6 weeks. The inset depicts a representative picture of the mammary glands. *, P < 0.05 compared to the corresponding weight of the β-cateninF/F implanted glands. C, Hemotoxlyn and Eoisin staining of mammary tissue of glands implanted β-catenin F/F and β-catenin−/− cells. Bars=200 μm.