Abstract 918: A key role for Crk adaptor proteins in basal breast cancer tumor growth in vivo

Abstract

Abstract Elevated levels of Crk proteins are observed in human cancers, including breast cancer, identifying a potential role for Crk in tumor progression. However, little is known about how Crk contributes to breast cancer progression in vivo. Crk proteins (CrkI, CrkII, CrkL) can regulate cell signaling downstream of integrins and various receptor tyrosine kinases through the formation of protein-protein complexes. We tested the hypothesis that Crk proteins are key signaling nodes for breast cancer tumorigenesis using shRNA-mediated knockdown of all 3 Crk proteins in highly aggressive human basal breast cancer cell lines. Crk knockdown cells show no differences in cell proliferation under 2D culture conditions, but show significantly decreased growth in soft agar under low serum conditions. This demonstrates that Crk loss imparts a renewed dependence on adhesion and growth factor signaling in highly aggressive breast tumor cell lines and highlights a requirement for Crk dependent signals for anchorage independent growth in vitro. Consistent with this, loss of Crk in basal breast cancer cells inhibited in vivo orthotopic tumor growth in nude mice. Using immunohistochemical staining of cells 3 days post-injection, Crk knockdown diminished proliferation in vivo, but did not alter cell apoptosis when compared to controls. This difference was reflected in the formation of smaller lesions by Crk knockdown cells by 8 days post-injection. Importantly, rescue of Crk protein expression restored in vivo tumor growth, demonstrating a specific requirement for Crk proteins in this process. To evaluate the significance of Crk in human breast cancer, immunohistochemistry was performed on two independent tissue microarrays of human breast tumors (n=209, n=234). Crk protein levels and the proliferative index were assessed by staining with antibodies to Crk and Ki67. Both Crk and Ki67 antigen positivity correlated strongly with high tumor grade (p=0.001, p=2.27e-10). Within both TMA datasets, CrkI/II (R2=0.3855, p=0.0004) and CrkL (R2=0.3845, p=0.0002) protein levels showed a strong positive correlation with cell proliferation within basal tumors, demonstrating a strong link between elevated Crk protein and an aggressive tumor phenotype. In addition to assessing Crk protein levels, a gene expression signature consisting of 151 genes was derived following Crk overexpression in a breast cancer cell line that initially had low levels of Crk. Notably, when this signature was applied to 5 breast cancer gene expression datasets (n=1469 breast cancers) there was a strong correlation with both the basal molecular subtype (p<6.7e-14), and with high tumor grade (p<6.4e-12), both of which are associated with poor prognosis, providing support for the IHC data. These findings demonstrate that Crk proteins are unexpectedly essential for growth of aggressive human breast cancer cell lines in vivo, and suggest a key role for Crk proteins in basal breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 918. doi:10.1158/1538-7445.AM2011-918