Cancer patients, including those with breast cancers, whose tumors are detected and treated early, often have good survival prognoses. If a tumor isn’t detected until it is well-developed or if it becomes resistant to therapy, however, then the chances for curative treatment are significantly reduced. For breast cancer patients, the most common cause of death is complications from tumor metastasis to distant organs, especially to the lung, brain, liver, and bone. Therefore, there is a substantial need for improved strategies to target metastatic disease. In a recent study published in Cancer Research, a research team led by Dr. Ygal Haupt at the University of Melbourne, Australia aimed to understand more clearly the molecular pathways involved in breast tumor metastasis. They describe a role for E6AP, an E3-ubiquitin ligase and factor in Rho-GTPase signaling pathways, in regulating cancer cell invasiveness and metastasis. Among their findings, they show that down-regulation or loss of E6AP expression in human breast tumors is associated with more aggressive disease. Further, restoring E6AP expression to a highly metastatic breast cancer cell line reduced metastasis and distant organ colonization in a mouse xenograft model. These data suggest that targeted therapies that augment E6AP expression or activity in humans may prevent breast cancer metastasis.
Previous work by Dr. Haupt’s group demonstrated an inverse association between E6AP protein expression and cancer aggressiveness – that is, invasive tumors expressed lower E6AP protein levels than normal breast tissue. In order to investigate the role of E6AP in breast cancer metastasis, Dr. Haupt’s team took a comprehensive approach that employed bioinformatic analyses of human tumors and in vitro and in vivo mouse models. To corroborate and characterize further the association between reduced E6AP expression and aggressive, metastatic disease, the researchers mined the METABRIC (Molecular Taxonomy of Breast Cancer International Consortium) database that compiles genomic and gene expression data with clinical outcomes for 1,992 patients from the United Kingdom and Canada. Their analyses supported their earlier report that reduced E6AP is associated with metastatic breast cancer, and showed further that reduced E6AP correlates with reduced survival and with aggressive triple-negative breast cancer (TNBC).
To investigated how modulating E6AP expression might affect tumor cell invasiveness in vitro. The Haupt team isolated mammary epithelial cells from both E6AP-deficient mice (B6.129S7-Ube3atm1Alb/J, Stock# 016590 (Ube3a encodes E6AP in mice)) and wild-type controls (C57BL/6J, Stock# 000664) and compared the cells’ abilities to invade endothelial cell cultures through a Matrigel layer. The E6AP-knockout epithelial cells were approximately three times more invasive than the wild-type cells. To more directly assess how E6AP expression affects metastasis, the researchers used a cell line xenograft system in which the human, highly metastatic, TNBC cell line MDA-MB-231-HM was engrafted into NSG™ mice (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ, Stock# 005557). MDA-MB-231-HM cells express lower levels of E6AP than other human breast cancer cell lines, but are more invasive. To examine how E6AP expression can modulate metastatic behavior of these cells, Dr. Haupt’s team created a variant of the cell line that expressed doxycycline (DOX)-inducible E6AP and that also constitutively expressed EGFP and luciferase to allow for facile imaging and tracking of the cells in vivo. The cell line was orthotopically engrafted into NSG™ female mammary gland, and the mice were treated subsequently with either DOX or vehicle. Primary tumor growth was similar in both engraftment groups. Metastasis was examined by resecting primary tumors from both groups when they reached 500 mm3, and then visualizing bioluminescence (BLI) at 10, 20, and 30 days after tumor resection. Luminescence intensity at both the resected tumor site and at sites in distant organs was reduced in the DOX-treated cohort, indicating that induced E6AP expression inhibited tumor cell migration and colonization to distant sites. DOX-treated mice also remained metastasis-free for longer compared to vehicle-treated controls. Lung colonization and overall engraftment rates were also reduced compared to controls in more direct metastasis experiments in which the DOX-inducible E6AP MDA-MB-231-HM cells were pre-treated with DOX to induce E6AP expression and then inoculated via tail vein injection into NSG™ mice.
E6AP is best known as an E3-ubiquitin ligase. To better understand the mechanism by which E6AP inhibits metastasis, the researchers employed a number of in vitro and biochemical assays. Their findings suggest that E6AP inhibits actin cytoskeleton remodeling by regulating the amount of ECT2 (epithelial cell transforming-2) protein that is targeted for proteasomal degradation. ECT2 is a guanine nucleotide exchange factor that is required for cytoskeleton-remodeling by RhoGTPases. When E6AP is expressed and active, ECT2 intracellular levels are reduced, and cytoskeleton dynamics are inhibited. Without the ability to alter their shape to support migration and infiltrative activity, E6AP-expressing tumor cells cannot metastasize.
This study identifies new target candidates for developing therapies to treat aggressive, metastatic tumors by more clearly defining the role of E6AP in cytoskeletal remodeling. Developing novel treatment paradigms that exploit these mechanisms could fill an urgent need for patients with advanced disease.
Mansour M et al. 2016. The E3-ligase E6AP represses breast cancer metastasis via regulation of ECT-Rho signaling. Cancer Research [Epub ahead of print], May 26, 2016. PMID: 27231202.