Abstract
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.
Highlights
Transducer of Cdc42-dependent actin assembly-1 (Toca-1) recruits actin regulatory proteins to invadopodia, and promotes breast tumor metastasis
We reported that Toca-1 expression levels are high in triple-negative breast cancer (TNBC) cell lines, which lack expression of estrogen receptor (ER)/progesterone receptor (PR)/human epidermal growth factor receptor 2 (HER2) receptors
Toca-1 levels correlate with p53 status in breast cancer cell lines We previously showed that Toca-1 levels were high in TNBC cell lines and a subset of invasive ductal carcinomas [11]
Summary
Transducer of Cdc42-dependent actin assembly-1 (Toca-1) recruits actin regulatory proteins to invadopodia, and promotes breast tumor metastasis. Metastasis is a complex process in which tumor cells acquire the ability to spread to other tissues via lymphatics or blood vessels. Invading cancer cells form filamentous actin (F-actin)-based membrane protrusions called invadopodia, whose extracellular matrix (ECM) degrading activity allows them to invade through basement membranes and migrate toward blood vessels [1]. Silencing of key components of invadopodia such as N-WASP or cortactin, leads to impaired tumor vascularization and Chander et al Breast Cancer Research (2014) 16:3413. We recently identified Toca-1 as a component of invadopodia in breast cancer cells, and that silencing of Toca-1 led to reduced incidence of metastasis to the lung in mammary orthotopic xenograft models [11]. We reported that Toca-1 expression levels are high in triple-negative breast cancer (TNBC) cell lines, which lack expression of estrogen receptor (ER)/progesterone receptor (PR)/human epidermal growth factor receptor 2 (HER2) receptors
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