The Ron receptor tyrosine kinase activates c-Abl to promote cell proliferation through tyrosine phosphorylation of PCNA in breast cancer.

Multiple growth pathways lead to enhanced proliferation in malignant cells. However, how the core machinery of DNA replication is regulated by growth signaling remains largely unclear. The sliding clamp proliferating cell nuclear antigen (PCNA) is an indispensable component of the DNA machinery responsible for replicating the genome and maintaining genomic integrity. We previously reported that epidermal growth factor receptor (EGFR) triggered tyrosine 211 (Y211) phosphorylation of PCNA, which in turn stabilized PCNA on chromatin to promote cell proliferation. Here we show that the phosphorylation can also be catalyzed by the non-receptor tyrosine kinase c-Abl. We further demonstrate that, in the absence of EGFR, signaling to PCNA can be attained through the activation of the Ron receptor tyrosine kinase and the downstream non-receptor tyrosine kinase c-Abl. We show that Ron and c-Abl form a complex, and that activation of Ron by its ligand, hepatocyte growth factor-like protein (HGFL), stimulates c-Abl kinase activity, which in turn directly phosphorylates PCNA at Y211 and leads to an increased level of chromatin-associated PCNA. Correspondingly, HGFL-induced Ron activation resulted in Y211 phosphorylation of PCNA while silencing of c-Abl blocked this effect. We show that c-Abl and Y211 phosphorylation of PCNA is an important axis downstream of Ron, which is required for cell proliferation. Treatment with a specific peptide that inhibits Y211 phosphorylation of PCNA or with the c-Abl pharmacological inhibitor imatinib suppressed HGFL-induced cell proliferation. Our findings identify the pathway of Ron-c-Abl-PCNA as a mechanism of oncogene-induced cell proliferation, with potentially important implications for development of combination therapy of breast cancer.

ß-Catenin is required for Ron receptor-induced mammary tumorigenesis.

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 ß-catenin expression and tyrosine phosphorylation. ß-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 ß-catenin and ß-catenin tyrosine phosphorylation downstream of Ron are not known. To investigate this association, we show that Ron and ß-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 ß-catenin, primarily on tyrosine residues Tyr 654 and Tyr 670. In addition, HGFL-mediated Ron activation induces both ß-catenin nuclear localization and transcriptional activity, with Tyr 654 and Tyr 670 residues of ß-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 ß-catenin-dependent transcriptional activation and cell growth, which can be rescued by activation of canonical Wnt/ß-catenin signaling. Moreover, we show that HGFL-dependent Ron activation mediates upregulation of the ß-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 ß-catenin. Finally, we show that genetic ablation of ß-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 ß-catenin is a crucial downstream regulator of Ron receptor activation and is an important mediator of mammary tumorigenesis.