Jalna Meens

The membrane cytoskeletal crosslinker ezrin is required for metastasis of breast carcinoma cells

IntroductionThe membrane cytoskeletal crosslinker ezrin participates in several functions including cell adhesion, motility and cell survival, and there is increasing evidence that it regulates tumour progression. However, the role played by ezrin in breast cancer metastasis has not been clearly delineated.MethodsWe examined the role of ezrin in metastasis using a highly metastatic murine mammary carcinoma cell line, namely AC2M2. Stable cell clones that overexpress wild-type ezrin or a dominant-negative amino-terminal domain of ezrin were selected. They were then tested for cell motility and invasion in vitro, and metastasis in a mouse in vivo tumour transplantation model.ResultsParental AC2M2 cells and cells overexpressing wild-type ezrin were transplanted into the mammary fat pad of syngeneic recipient mice; these animals subsequently developed lung metastases. In contrast, expression of the dominant-negative amino-terminal ezrin domain markedly inhibited lung metastasis. Consistent with this effect, we observed that the expression of amino-terminal ezrin caused strong membrane localization of cadherin, with increased cell–cell contact and a decrease in cell motility and invasion, whereas cells expressing wild-type ezrin exhibited strong cytoplasmic expression of cadherins and pseudopodia extensions. In addition, inhibitors of phosphatidylinositol 3-kinase and c-Src significantly blocked cell motility and invasion of AC2M2 cells expressing wild-type ezrin. We further found that overexpression of amino-terminal ezrin reduced levels of Akt pS473 and cytoskeletal-associated c-Src pY418 in AC2M2 cells, which contrasts with the high levels of phosphorylation of these proteins in cells expressing wild-type ezrin. Phosphorylated Erk1/2 was also reduced in amino-terminal ezrin expressing cells, although a mitogen-activated protein kinase kinase (MEK) inhibitor had no detectable effect on cell motility or invasion in this system.ConclusionOur findings indicate that ezrin is required for breast cancer metastasis, and that c-Src and phosphatidylinositol 3-kinase/Akt are effectors of ezrin in the cell motility and invasion stages of the metastatic process. Together, these results suggest that blocking ezrin function may represent a novel and effective strategy for preventing breast cancer metastasis.

A Novel RET Kinase–β-Catenin Signaling Pathway Contributes to Tumorigenesis in Thyroid Carcinoma

The RET receptor tyrosine kinase has essential roles in cell survival, differentiation, and proliferation. Oncogenic activation of RET causes the cancer syndrome multiple endocrine neoplasia type 2 (MEN 2) and is a frequent event in sporadic thyroid carcinomas. However, the molecular mechanisms underlying RETs potent transforming and mitogenic signals are still not clear. Here, we show that nuclear localization of beta-catenin is frequent in both thyroid tumors and their metastases from MEN 2 patients, suggesting a novel mechanism of RET-mediated function through the beta-catenin signaling pathway. We show that RET binds to, and tyrosine phosphorylates, beta-catenin and show that the interaction between RET and beta-catenin can be direct and independent of cytoplasmic kinases, such as SRC. As a result of RET-mediated tyrosine phosphorylation, beta-catenin escapes cytosolic down-regulation by the adenomatous polyposis coli/Axin/glycogen synthase kinase-3 complex and accumulates in the nucleus, where it can stimulate beta-catenin-specific transcriptional programs in a RET-dependent fashion. We show that down-regulation of beta-catenin activity decreases RET-mediated cell proliferation, colony formation, and tumor growth in nude mice. Together, our data show that a beta-catenin-RET kinase pathway is a critical contributor to the development and metastasis of human thyroid carcinoma.

Src and FAK mediate cell–matrix adhesion‐dependent activation of met during transformation of breast epithelial cells

Cell–matrix adhesion has been shown to promote activation of the hepatocyte growth factor receptor, Met, in a ligand‐independent manner. This process has been linked to transformation and tumorigenesis in a variety of cancer types. In the present report, we describe a key role of integrin signaling via the Src/FAK axis in the activation of Met in breast epithelial and carcinoma cells. Expression of an activated Src mutant in non‐neoplastic breast epithelial cells or in carcinoma cells was found to increase phosphorylation of Met at regulatory tyrosines in the auto‐activation loop domain, correlating with increased cell spreading and filopodia extensions. Furthermore, phosphorylated Met is complexed with β1 integrins and is co‐localized with vinculin and FAK at focal adhesions in epithelial cells expressing activated Src. Conversely, genetic or pharmacological inhibition of Src abrogates constitutive Met phosphorylation in carcinoma cells or epithelial cells expressing activated Src, and inhibits filopodia formation. Interestingly, Src‐dependent phosphorylation of Met requires cell–matrix adhesion, as well as actin stress fiber assembly. Phosphorylation of FAK by Src is also required for Src‐induced Met phosphorylation, emphasizing the importance of the Src/FAK signaling pathway. However, stimulation of Met phosphorylation by addition of exogenous HGF in epithelial cells is refractory to inhibition of Src family kinases, indicating that HGF‐dependent and Src/integrin‐dependent Met activation occur via distinct mechanisms. Together these findings demonstrate a novel mechanism by which the Src/FAK axis links signals from the integrin adhesion complex to promote Met activation in breast epithelial cells. J. Cell. Biochem. 107: 1168–1181, 2009.

Fer Protein-Tyrosine Kinase Promotes Lung Adenocarcinoma Cell Invasion and Tumor Metastasis

Epidermal growth factor receptor (EGFR) is frequently amplified or mutated in non–small cell lung cancer (NSCLC). Although Fer protein-tyrosine kinase signals downstream of EGFR, its role in NSCLC tumor progression has not been reported. Here, Fer kinase was elevated in NSCLC tumors compared to normal lung epithelium. EGFR signaling in NSCLC cells fosters rapid Fer activation and increased localization to lamellipodia. Stable silencing of Fer in H1299 lung adenocarcinoma cells (Fer KD) caused impaired EGFR-induced lamellipodia formation compared to control cells. Fer KD NSCLC cells showed reduced Vav2 tyrosine phosphorylation that was correlated with direct Fer-mediated phosphorylation of Vav2 on tyrosine-172, which was previously reported to increase the guanine nucleotide exchange factor activity of Vav2. Indeed, Fer KD cells displayed defects in Rac-GTP localization to lamellipodia, cell migration, and cell invasion in vitro. To test the role of Fer in NSCLC progression and metastasis, control and Fer KD cells were grown as subcutaneous tumors in mice. Although Fer was not required for tumor growth, Fer KD tumor-bearing mice had significantly fewer numbers of spontaneous metastases. Combined, these data demonstrate that Fer kinase is elevated in NSCLC tumors and is important for cellular invasion and metastasis. Implications: Fer protein-tyrosine kinase is a potential therapeutic target in metastatic lung cancer. Mol Cancer Res; 11(8); 952–63. ©2013 AACR.

Transducer of Cdc42-dependent actin assembly promotes breast cancer invasion and metastasis.

Metastatic breast adenocarcinomas display activation signatures for signaling pathways that trigger cell motility and tissue invasion. Here, we report that the adaptor protein transducer of Cdc42-dependent actin assembly-1 (Toca-1) is expressed in highly invasive breast cancers and regulates their metastatic phenotypes. We show that Toca-1 localizes to the filamentous actin-rich core of invadopodial protrusions actively degrading the extracellular matrix (ECM). Toca-1 colocalizes with Cortactin, and we show that this interaction is mediated by the SH3 domain of Toca-1. Stable knockdown (KD) of Toca-1 expression in MDA-MB-231 cells led to a significant defect in epidermal growth factor (EGF)-induced cell migration and invasion. Toca-1 KD cells also showed significant defects in EGF- and Src-induced ECM digestion and formation of invadopodial membrane protrusions. To test the role of Toca-1 in metastasis, we achieved stable Toca-1 KD in both human and rat metastatic breast adenocarcinoma cell lines. Orthotopic tumor xenografting of control and Toca-1 KD cells in natural-killer /B-/T-cell-deficient mice revealed a significant defect in spontaneous lung metastases with Toca-1 silencing in vivo. In contrast, no defects in primary tumor growth or lung seeding following tail vein injection of Toca-1 KD cells was observed, suggesting that Toca-1 functions at an early step in the dissemination of metastatic breast tumor cells. Taken together, our results identify Toca-1 as a proinvasive protein in breast adenocarcinoma and a potential therapeutic target to limit tumor metastasis.

CIP4 promotes lung adenocarcinoma metastasis and is associated with poor prognosis

Aberrant epidermal growth factor receptor (EGFR) signaling in non-small cell lung cancer (NSCLC) is linked to tumor progression, metastasis and poor survival rates. Here we report the role of Cdc42-interacting protein 4 (CIP4) in the regulation of NSCLC cell invasiveness and tumor metastasis. CIP4 was highly expressed in a panel of NSCLC cell lines and normal lung epithelial cell lines. Stable knockdown (KD) of CIP4 in lung adenocarcinoma H1299 cells, expressing wild-type EGFR, led to increased EGFR levels on the cell surface and defects in sustained activation of Erk kinase in H1299 cells treated with EGF. CIP4 localized to leading edge projections in NSCLC cells, and CIP4 KD cells displayed defects in EGF-induced cell motility and invasion through extracellular matrix. This correlated with reduced expression and activity of matrix metalloproteinase-2 (MMP-2) in CIP4 KD cells compared with control. In xenograft assays, CIP4 silencing had no effect on tumor growth but resulted in significant defects in spontaneous metastases to the lungs from these subcutaneous tumors. This correlated with reduced expression of the Erk target gene Zeb1 and the Zeb1 target gene MMP-2 in CIP4 KD tumors compared with control. CIP4 also enhanced rates of metastasis to the liver and lungs in an intrasplenic experimental metastasis model. In human NSCLC tumor sections, CIP4 expression was elevated greater than or equal to twofold in 43% of adenocarcinomas and 32% of squamous carcinomas compared with adjacent normal lung tissues. Analysis of microarray data for NSCLC patients also revealed that high CIP4 transcript levels correlated with reduced overall survival. Together, these results identify CIP4 as a positive regulator of NSCLC metastasis and a potential poor prognostic biomarker in lung adenocarcinoma.

Endophilin A2 Promotes TNBC Cell Invasion and Tumor Metastasis

Triple-negative breast cancers (TNBCs) are highly aggressive cancers that lack targeted therapies. However, EGFR is frequently activated in a subset of TNBCs and represents a viable clinical target. Because the endocytic adaptor protein Endophilin A2 (SH3GL1/Endo II) has been implicated in EGFR internalization, we investigated Endo II expression and function in human TNBCs. Endo II expression was high in several TNBC cells compared with normal breast epithelial cells. Stable knockdown (KD) of Endo II was achieved in two TNBC cell lines, and although cell viability was unaffected, defects in receptor-mediated endocytosis were observed. EGFR signaling to Erk and Akt kinases was impaired in Endo II KD cells, and this correlated with reduced rates of EGFR internalization and cell motility. Endo II KD cells also displayed defects in three dimensional (3D) cell invasion, and this correlated with impaired extracellular matrix degradation and internalization of MT1-MMP. Endo II silencing also caused a significant reduction in TNBC tumor growth and lung metastasis in mammary orthotopic tumor xenograft assays. In human breast tumor specimens, Endo II expression was highest in TNBC tumors compared with other subtypes, and at the level of gene expression, high Endo II was associated with reduced relapse-free survival in patients with basal-like breast cancers. Together, these results identify a positive role for Endo II in TNBC tumor metastasis and a potential link with poor prognosis. Implications: Endophilin A2 and related adaptor proteins represent important signaling hubs to target in metastatic cancers. Mol Cancer Res; 13(6); 1044–55. ©2015 AACR.

Toca-1 is suppressed by p53 to limit breast cancer cell invasion and tumor metastasis

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.

Abstract 3796: Endophilin II promotes breast tumor progression and metastasis.

Invadopodia are filamentous actin-based membrane protrusions with extracellular matrix (ECM) degrading activity that are implicated in tumor progression and metastasis. A key step in invadopodia formation involves the recruitment of the membrane type 1 matrix metalloprotease (MT1-MMP) to invadopodia precursors. Surface expression of MT1-MMP promotes ECM degradation and cell invasiveness, and this is dependent on Src kinase-induced phosphorylation and inhibition of endocytic proteins. In fibroblasts, MT1-MMP internalization was regulated by Endophilin II (Endo II), a widely expressed protein implicated in synaptic vesicle uncoating and recycling in neurons. Here, we tested the role of Endo II in invadopodia formation in basal-like breast cancer cells, and in tumor progression and metastasis in mice. High levels of Endo II expression were observed in basal breast cancer cell lines with high Src kinase and ECM-degrading activity. Immunohistochemistry staining also identified Endo II expression in primary breast tumors and lymph node metastases. To define the functions of Endo II, we achieved stable silencing of Endo II in two basal breast cancer cell lines (MDA-MB-231, HCC1806). Although Endo II was not required for uptake of receptors via clathrin-mediated endocytosis (EGFR, TfR), we observed a significant reduction in numbers of invadopodia and ECM degradation upon Endo II silencing. Consistent with invadopodia defects in vitro, Endo II silencing led to a significant reduction in tumor growth and metastases in mouse orthotopic tumor xenografts assays performed with both basal breast cancer cell models. This study provides novel insights into roles of Endo II as a positive regulator of invadopodia and basal breast cancer metastasis. Citation Format: Tomas Baldassarre, Jalna Meens, Andrew W. Craig. Endophilin II promotes breast tumor progression and metastasis. [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 3796. doi:10.1158/1538-7445.AM2013-3796

Abstract 5311: Transducer of Cdc42-dependent actin assembly-1 (Toca-1) promotes breast cancer invasion and metastasis

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