Elevated p130Cas (Crk-associated substrate) levels are found in aggressive breast tumors and are associated with poor prognosis and resistance to standard therapeutics in patients. p130Cas signals majorly through its phosphorylated substrate domain (SD) that contains 15 tyrosine motifs (YxxP) which recruit effector molecules. Tyrosine phosphorylation of p130Cas is important for mediating migration, invasion, tumor promotion, and metastasis. We previously developed a Src*/SD fusion molecule approach, where the SD is constitutively phosphorylated. In a polyoma middle T-antigen (PyMT)/Src*/SD double-transgenic mouse model, Src*/SD accelerates PyMT-induced tumor growth and promotes a more aggressive phenotype. To test whether Src*/SD also drives metastasis and which of the YxxP motifs are involved in this process, full-length and truncated SD molecules fused to Src* were expressed in breast cancer cells. The functionality of the Src*/SD fragments was analyzed in vitro, and the active proteins were tested in vivo in an orthotopic mouse model. Breast cancer cells expressing the full-length SD and the functional smaller SD fragment (spanning SD motifs 6–10) were injected into the mammary fat pads of mice. The tumor progression was monitored by bioluminescence imaging and caliper measurements. Compared with control animals, the complete SD promoted primary tumor growth and an earlier onset of metastases. Importantly, both the complete and truncated SD significantly increased the occurrence of metastases to multiple organs. These studies provide strong evidence that the phosphorylated p130Cas SD motifs 6–10 (Y236, Y249, Y267, Y287, and Y306) are important for driving mammary carcinoma progression.
p130Cas Breast cancer Substrate domain Src*/SD Metastasis Bioluminescence imaging Mouse tumor model
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We gratefully acknowledge Joan Massague and Robert I. Nicholson for cell lines and Amy H. Bouton for the CasB antibody. We thank Matthew D. Layne for critical reading of the manuscript. We greatly appreciate the help of Manish Bais and Tom Balon with bioluminescence imaging and of Kim Bayer in establishing the animal procedures. All bioluminescence imaging was performed at the IVIS Imaging Core of Boston University School of Medicine. This work was supported by the National Cancer Institute grant CA106468, by the National Center for Advancing Translational Sciences through the BU-CTSI grant U54TR001012, both from the National Institute of Health, and the Susan G. Komen for the Cure Breast Cancer Foundation grant KG101208.
Compliance with ethical standards
Animal experiments were performed and the mice euthanized according to guidelines of and approved by the Institutional Animal Care and Use Committee (IACUC) at Boston University.
Cell morphology of LM2 cells expressing Src*/SD and control constructs that did not induce significant morphological changes. Cells (A and B) Cells (100,000 cells/well in 6-well plates) stably transduced with the indicated inducible constructs were treated for 24 h with DOX or left untreated. KM, SrcKM; *, Src*. c, control (empty vector). Left panels, expression (anti-HA) and phosphorylation (p-Tyr) of the fusion molecules in WCE (30 μg) was confirmed by IB. n.s., non-specific. Right panels, cell morphology was analyzed by microscopy. Scale, 100 μm. (GIF 212 kb)
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