Frequent overexpression of AMAP1, an Arf6 effector in cell invasion, is characteristic of the MMTV-PyMT rather than the MMTV-Neu human breast cancer model

Yutaro Otsuka, Tsukasa Oikawa, Hinako Yoshino, Shigeru Hashimoto, Haruka Handa, Hiroki Yamamoto, Ari Hashimoto, Hisataka Sabe

The small GTPase Arf6 and its downstream effector AMAP1 (also called ASAP1/DDEF1) constitute a signaling pathway promoting cell invasion, in which AMAP1 interacts with several different proteins, including PRKD2, EPB41L5, paxillin, and cortactin. Components of this pathway are often overexpressed in human breast cancer cells, to be correlated with poor prognosis of the patients, whereas overexpression of the Arf6 pathway did not correlate with the four main molecular classes of human breast tumors. In this pathway, receptor tyrosine kinases, including EGFR and Her2, activate Arf6 via GEP100. MMTV-PyMT mice and MMTV-Neu mice are well-established models of human breast cancer, and exhibit the early dissemination and the lung metastasis, by utilizing protein tyrosine phosphorylation for oncogenesis. PyMT-tumors and Neu-tumors are known to have overlapping gene expression profiles, which primarily correspond to the luminal B-type of human mammary tumors, although they differ in the time necessary for tumor onset and metastasis. Given the common usage of protein tyrosine phosphorylation, as well as the frequent use of these animal models for studying breast cancer at the molecular level, we here investigated whether mammary tumors in these mouse models utilize the Arf6-based pathway for invasion. Expression levels of Arf6, AMAP1, and GEP100 were analyzed in PyMT-tumors and Neu-tumors by western blotting. Expression of Arf6 and AMAP1 was also analyzed by immunohistochemistry. The involvement of AMAP1 in invasion, and the possible correlation of its high expression levels with cancer mesenchymal properties were also investigated. We found that PyMT-tumors, but not Neu-tumors, frequently overexpress AMAP1 and use it for invasion, whereas both types of tumors expressed Arf6 and GEP100 at different levels. High levels of the AMAP1 expression among PyMT-tumor cells were frequently correlated with loss of the epithelial marker CK8 and also with expression of the mesenchymal marker vimentin both at the primary sites and at sites of the lung metastases. PyMT-tumors appear to frequently utilize the Arf6-based invasive machinery, whereas Neu-tumors do not. Our results suggest that MMTV-PyMT mice, rather than MMTV-Neu mice, are useful to study the Arf6-based mammary tumor malignancies, as a representative model of human breast cancer.