Contributions of the RhoA guanine nucleotide exchange factor Net1 to polyoma middle T antigen-mediated mammary gland tumorigenesis and metastasis

Abstract

Background: The RhoA activating protein Net1 contributes to breast cancer cell proliferation, motility, and invasion in vitro, yet little is known about its roles in mammary gland tumorigenesis and metastasis. Methods: Net1 knockout (KO) mice were bred to mice with mammary gland specific expression of the polyoma middle T antigen (PyMT) oncogene. Mammary gland tumorigenesis and lung metastasis were monitored. Individual tumors were assessed for proliferation, apoptosis, angiogenesis, RhoA activation, and activation of PyMT-dependent signaling pathways. Primary tumor cells from wild-type and Net1 KO mice were transplanted into the mammary glands of wild-type, nontumor-bearing mice, and tumor growth and metastasis were assessed. Gene expression in wild-type and Net1 KO tumors was analyzed by gene ontology enrichment and for relative activation of gene expression signatures indicative of signaling pathways important for breast cancer initiation and progression. A gene expression signature indicative of Net1 function was identified. Human breast cancer gene expression profiles were screened for the presence of a Net1 gene expression signature. Results: We show that Net1 makes fundamental contributions to mammary gland tumorigenesis and metastasis. Net1 deletion delays tumorigenesis and strongly suppresses metastasis in PyMT-expressing mice. Moreover, we observe that loss of Net1 reduces cancer cell proliferation, inhibits tumor angiogenesis, and promotes tumor cell apoptosis. Net1 is required for maximal RhoA activation within tumors and for primary tumor cell motility. Furthermore, the ability of PyMT to initiate oncogenic signaling to ERK1/2 and PI3K/Akt1 is inhibited by Net1 deletion. Primary tumor cell transplantation indicates that the reduction in tumor angiogenesis and lung metastasis observed upon Net1 deletion are tumor cell autonomous effects. Using a gene expression signature indicative of Net1 activity, we show that Net1 signaling is activated in 10% of human breast cancers, and that this correlates with elevated proliferation and PI3K pathway activity. We also demonstrate that human breast cancer patients with a high Net1 gene expression signature experience shorter distant metastasis-free survival. Conclusions: These data indicate that Net1 is required for tumor progression in the PyMT mouse model and suggest that Net1 may contribute to breast cancer progression in humans.