P2-02-02: Mutation of the APC Tumor Suppressor Stimulates Breast Cancer Cell Proliferation through Hyperactivation of FAK/Src/JNK Signaling.

Abstract

Abstract Background: The Adenomatous Polyposis Coli (APC) tumor suppressor gene is silenced by hypermethylation or mutated in as many as 70% of human sporadic breast cancers. In mouse models, germline mutation of Apc disrupts normal mammary development and predisposes to mammary tumor formation. In an effort to more accurately model human breast cancer and determine the role for APC inactivation in the context of other oncogenic events, our laboratory has recently demonstrated that APC loss promotes mammary tumor development in the presence of the Polyoma virus middle T antigen (PyMT) oncogene, and results in a tumor phenotype resembling human metaplastic breast cancer. In this model, Apc mutation significantly enhances tumor cell proliferation but does not impact Wnt/β-catenin signaling. Because APC loss also induces mammary tumor cell spreading and morphological changes, we hypothesized that APC may regulate proliferation via signaling by the focal adhesion kinase (FAK) and Src (kinases) downstream of altered cell-matrix interactions. Materials and Methods: Cell lines were established from primary cultures of experimental (MMTV-PyMT;ApcMin/+) and control (MMTV-PyMT;Apc+/+) mammary tumors and maintained in RPMI 1640 supplemented with 10% FBS and antibiotics at 37°C with 5% CO2. Cell proliferation was measured by incorporation of 5-bromo-2-deoxyuridine (BrdU), and subsequent immunofluorescence (IF) with an anti-BrdU antibody. Western blot analysis and IF were utilized to quantify the expression of junctional and signaling molecules and determine their localization, respectively. To investigate Wnt/β-catenin pathway activation, dual-luciferase reporter assays were performed using cells transfected with either Top-Flash or Fop-Flash reporter constructs. Results: Cell lines isolated from mammary tumors from MMTV-PyMT;ApcMin/+ mice had altered morphology and increased rates of proliferation, both of which are consistent with our previous in vivo findings using this mouse model. In vitro, Apc mutation was associated with increased phosphorylation of FAK, Src and Jun kinase (JNK). Activation of both Src and JNK were required for the enhanced proliferation in the MMTV-PyMT;ApcMin/+ cell lines. In contrast, we did not observe any change in the expression of Wnt/β-catenin target genes, β-catenin subcellular localization, or β-catenin/TCF-mediated transcriptional activity. Combined, these data suggest that the proliferative and morphological changes mediated by APC loss in the PyMT breast cancer model are dependent on enhanced signaling downstream of cell-matrix interactions but do not involve the Wnt/β-catenin pathway. Discussion: These data indicate that APC status is an important determinant of breast cancer cell behavior and specifically suggest that integrin-mediated signaling networks, including FAK, Src and JNK, are hyperactivated by APC loss to promote tumor cell proliferation. Moreover, these preclinical model systems offer a platform for further dissecting the molecular mechanisms by which APC contributes to breast carcinogenesis as well as identifying therapeutic strategies that may be effective in APC-deficient breast cancers. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-02-02.