Abstract
BackgroundThe 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.MethodsNet1 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.ResultsWe 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.ConclusionsThese 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.
Highlights
The RhoA activating protein neuroepithelial transforming gene 1 (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
In the present work we demonstrate that Net1 is critical for mammary gland tumorigenesis and metastasis in the mouse mammary tumor virus (MMTV)-polyoma middle T antigen (PyMT) mouse genetic model of breast cancer, and demonstrate obligate signaling pathways that are regulated by Net1
Net1 is required for tumorigenesis and metastasis in MMTV-PyMT mice We have shown previously that mice lacking Net1 are healthy, but experience a short delay in mammary gland development during puberty characterized by reduced estrogen receptor alpha (ERα) expression, reduced proliferation, and less ductal branching
Summary
The RhoA activating protein Net contributes to breast cancer cell proliferation, motility, and invasion in vitro, yet little is known about its roles in mammary gland tumorigenesis and metastasis. As regulators of cell proliferation, cytoskeletal organization, and cell motility, Rho GTPases are essential to dissemination of cancer cells throughout the body. The Rho GTPase family consists of 20 genes in humans, with Cdc, Rac, and RhoA being the most thoroughly studied [1, 2]. RhoA, and RhoC are commonly overexpressed in human breast cancers and RNAimediated knockdown or deletion of these genes inhibits tumorigenesis and metastasis [3,4,5,6,7]. Rac and RhoA/C contribute to metastasis by regulating separate types of invasive behavior in cancer cells, with Rac driving integrin-dependent, mesenchymal-type movement and RhoA/C driving integrin-independent, amoeboid movement. Cancer cells switch between these types of movement depending on the extracellular obstacles they are traversing, and inhibition of either of these forms of movement significantly inhibits invasive activity [8,9,10]
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