Activated platelets promote the proliferation and metastatic potential of cancer cells. Platelet activation is largely mediated through ADP engagement of purinergic P2Y12 receptors on platelets. We examined the potential of the reversible P2Y12 inhibitor ticagrelor, an agent used clinically to prevent cardiovascular and cerebrovascular events, to reduce tumor growth and metastasis. In vitro, MCF-7, MDA-MB-468, and MDA-MB-231 human mammary carcinoma cells exhibited decreased interaction with platelets treated with ticagrelor compared to untreated platelets. Prevention of tumor cell-platelet interactions through pretreatment of platelets with ticagrelor did not improve natural killer cell-mediated tumor cell killing of K562 myelogenous leukemia target cells. Additionally, ticagrelor had no effect on proliferation of 4T1 mouse mammary carcinoma cells co-cultured with platelets, or on primary 4T1 tumor growth. In an orthotopic 4T1 breast cancer model, ticagrelor (10 mg/kg), but not clopidogrel (10 mg/kg) or saline, resulted in reduced metastasis and improved survival. Ticagrelor treatment was associated with a marked reduction in tumor cell-platelet aggregates in the lungs at 10, 30 and 60 min post-intravenous inoculation. These findings suggest a role for P2Y12-mediated platelet activation in promoting metastasis, and provide support for the use of ticagrelor in the prevention of breast cancer spread.

IntroductionTransducer of Cdc42-dependent actin assembly-1 (Toca-1) recruits actin regulatory proteins to invadopodia, and promotes breast tumor metastasis. Since metastatic breast tumors frequently harbor mutations in the tumor suppressor p53, we tested whether p53 regulates Toca-1 expression.MethodsNormal mammary epithelial cells (HBL-100, MCF10A) and breast cancer cell lines expressing wild-type (WT) p53 (DU4475, MTLn3) were treated with camptothecin or Nutlin-3 to stabilize p53 to test effects on Toca-1 mRNA and protein levels. Chromatin immunoprecipitation (ChIP) assays were performed to identify p53 binding site in Toca-1 gene. Stable silencing of p53 and Toca-1 were performed in MTLn3 cells to test effects on invadopodia and cell invasion in vitro, and tumor metastasis in vivo.ResultsWe observed that breast cancer cell lines with mutant p53 have high levels of Toca-1 compared to those with WT p53. Stabilization of WT p53 led to further reduction in Toca-1 mRNA and protein levels in normal breast epithelial cells and breast cancer cells. ChIP assays revealed p53 binding within intron 2 of toca1, and reduced histone acetylation within its promoter region upon p53 upregulation or activation. Stable silencing of WT p53 in MTLn3 cells led to increased extracellular matrix degradation and cell invasion compared to control cells. Interestingly, the combined silencing of p53 and Toca-1 led to a partial rescue of these effects of p53 silencing in vitro and reduced lung metastases in mice. In human breast tumors, Toca-1 levels were high in subtypes with frequent p53 mutations, and high Toca-1 transcript levels correlated with increased risk of relapse.ConclusionsBased on these findings, we conclude that loss of p53 tumor suppressor function in breast cancers leads to upregulation of Toca-1, and results in enhanced risk of developing metastatic disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0503-x) contains supplementary material, which is available to authorized users.

Transducer of Cdc42-dependent actin assembly-1 (Toca-1) is an adaptor protein that recruits key actin regulatory proteins to invadopodia in metastatic breast cancer cells. We recently showed that stable silencing of Toca-1 led to reduced invadopodia formation, cell invasion and tumor metastasis in an orthotopic xenograft model in mice. These results raise the possibility that Toca-1 upregulation may occur during tumor progression to metastatic disease. Here we show that Toca-1 expression is higher in triple negative breast cancers (HER2/ER/PR) compared to other subtypes. Since triple negative breast cancers frequently harbor mutations in the tumor suppressor p53, we tested whether p53 regulates Toca-1 expression. In wild-type (WT) p53-expressing normal breast epithelial and breast cancer cells, stabilization of WT p53 by inducing DNA damage, or treating cells with Mdm2 inhibitor Nutlin-3, led to dose-dependent reductions in Toca-1 mRNA and protein expression. Likewise, ectopic expression of WT p53 in p53-null HCC1806 cells also led to repression of Toca-1 levels. To test if this was a direct effect of p53 on the Toca-1 gene, chromatin immunoprecipitation (ChIP) studies were performed. Indeed, the proximal promoter region and intron 2 of the Toca-1 gene (which contained a predicted p53 binding site) were detected in p53 ChIP assays in WT p53-expressing cells, but not mutant p53 expressing breast cancer cells. Together, these results identify a novel pathway to inhibit Toca-1 expression in cells with WT p53, and imply that the loss of p53 in highly metastatic breast cancer subtypes may lead to elevated Toca-1 expression and progression to metastatic disease. Citation Format: Harish Chander, Colin D. Brien, Peter Truesdell, Doris Germain, Andrew W. B. Craig. Tumor suppressor p53 inhibits expression of the pro-metastasis protein Toca-1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 787. doi:10.1158/1538-7445.AM2013-787

Metastatic breast adenocarcinomas display activation signatures for signaling pathways like Epidermal Growth Factor Receptor (EGFR) that trigger cell motility and tissue invasion. Transducer of Cdc42-dependent actin assembly-1 (Toca-1) is an adaptor protein that promotes recruitment of actin nucleation promoting factors to cellular membranes. Although these actin regulatory proteins have been linked to breast cancer invasion, the role of Toca-1 in this process has not been reported. In this study, breast cancer cell lines with a range of invasive and metastatic properties were used, including human MDA-MB-231 cells, rat MTLn3 cells, and MTLn3-B1 cells expressing human EGFR. To test the role of Toca-1 in these cell lines, we established stable Toca-1 knock-down (KD) using lentiviral delivery of shRNAs specific for human or rat Toca-1. Here, we report that Toca-1 is expressed in highly invasive breast cancers cell lines, and that Toca-1 co-localizes with components of invadopodia. Toca-1 localizes to the filamentous actin-rich core of invadopodial protrusions in breast cancer cells that are actively degrading the extracellular matrix (ECM). In MDA-MB-231 cells, Toca-1 KD led to a significant defect in EGF-induced cell migration and invasion compared to control cells. This correlated with a significant defect in EGF- and Src-induced ECM digestion in Toca-1 KD compared to control MDA-MB-231 cells. Next, control and Toca-1 KD cells were compared in orthotopic mammary tumors that were xenografted in Rag2−/−:[[Unsupported Character - Symbol Font ]]c−/− mice. Although Toca-1 KD remained effective in primary tumors, no defects in tumor growth were observed. However, mice bearing Toca-1 KD cell tumors displayed a significant reduction in spontaneous metastases to the lung. Similar results were obtained for weakly metastatic MDA-MB-231 cells and highly metastatic MTLn3-B1 cells. In contrast, Toca-1 was not required for efficient lung seeding following tail vein injection of MTLn3-B1 cells, suggesting that Toca-1 functions at an early step in the dissemination of metastatic breast tumor cells. Taken together, this investigation identifies Toca-1 as a pro-invasive protein in breast cancer, and a potential therapeutic target to limit tumor metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5311. doi:1538-7445.AM2012-5311

Invadopodia are extracellular matrix (ECM)-degrading structures that promote tissue invasion and metastasis of tumor cells. A family of adaptor proteins with F-BAR and SH3 domains, including FBP17 and Toca-1, have been identified as key scaffolds for recruiting actin regulatory proteins (eg. Cdc42, formins, N-WASP, dynamin) to promote formation of invadopodia in bladder and breast cancers, respectively. In this study, we investigated the potential role of FBP17 in breast cancer models. We observed FBP17 expression in normal breast epithelial cells (MCF10A), and at higher levels in basal breast cancer cell lines (MDA-MB-231) compared to luminal cell lines. Since basal breast cancers frequently harbor mutations in tumor suppressor p53, we tested whether wild-type (WT) p53 regulates expression of FBP17, which may explain higher expression in basal breast cancers. Camptothecin (CPT) treatment of WT p53-expressing breast cancer cells (MCF-7, DU4475) led to a dose dependent reduction in FBP17 mRNA and protein levels, and this was inversely correlated with p53 levels or its target p21WAF1. The downregulation of FBP17 was also observed using Nutlin-3, an Mdm2 inhibitor, indicating that WT p53 activation by multiple methods causes a rapid downregulation of FBP17. Currently we are testing whether this is a direct or indirect effect of p53 on FBP17 gene expression. As expected, FBP17 formed complexes with actin regulatory proteins (dynamin, cortactin, Arp2/3) that accumulate and promote invadopodia formation in MDA-MB-231 cells. Stable silencing of FBP17 in these cells led to a significant reduction in ECM degradation. This is consistent with FBP17 promoting invadopodia formation. Tumor xenograft models are in progress to determine whether FBP17 also promotes tumor metastasis. FBP17 expression levels in human breast tumors are currently being tested to determine whether altered expression occurs in molecular subtypes with frequent mutations in p53 (HER2, basal) and in their metastases. Overall, these results are consistent with FBP17 participating in cancer cell invasion, and supports further studies of its role in cancer metastasis. Citation Format: Harish Chander, Kathleen Watts, Justin Pogmore, Peter Truesdell, Colin Brien, Andrew W B Craig. Formin-binding protein-17 (FBP17) is a target of p53 and promotes invadopodia formation in breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4053. doi:10.1158/1538-7445.AM2014-4053