MENA Confers Resistance to Paclitaxel in Triple-Negative Breast Cancer

Taxane therapy remains the standard of care for triple-negative breast cancer. However, high frequencies of recurrence and progression in treated patients indicate that metastatic breast cancer cells can acquire resistance to this drug. The actin regulatory protein MENA and particularly its invasive isoform, MENAINV , are established drivers of metastasis. MENAINV expression is significantly correlated with metastasis and poor outcome in human patients with breast cancer. We investigated whether MENA isoforms might play a role in driving resistance to chemotherapeutics. We find that both MENA and MENAINV confer resistance to the taxane paclitaxel, but not to the widely used DNA-damaging agents doxorubicin or cisplatin. Furthermore, paclitaxel treatment does not attenuate growth of MENAINV -driven metastatic lesions. Mechanistically, MENA isoform expression alters the ratio of dynamic and stable microtubule populations in paclitaxel-treated cells. MENA expression also increases MAPK signaling in response to paclitaxel treatment. Decreasing ERK phosphorylation by cotreatment with MEK inhibitor restored paclitaxel sensitivity by driving microtubule stabilization in MENA isoform-expressing cells. Our results reveal a novel mechanism of taxane resistance in highly metastatic breast cancer cells and identify a combination therapy to overcome such resistance

In vivo postnatal electroporation and time-lapse imaging of neuroblast migration in mouse acute brain slices.

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration. In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair

Characterization of the expression of the pro-metastatic MenaINV isoform during breast tumor progression

Several functionally distinct isoforms of the actin regulatory Mena are produced by alternative splicing during tumor progression. Forced expression of the Mena[superscript INV] isoform drives invasion, intravasation and metastasis. However, the abundance and distribution of endogenously expressed Mena[superscript INV] within primary tumors during progression remain unknown, as most studies to date have only assessed relative mRNA levels from dissociated tumor samples. We have developed a Mena[superscript INV] isoform-specific monoclonal antibody and used it to examine Mena[superscript INV]expression patterns in mouse mammary and human breast tumors. Mena[superscript INV] expression increases during tumor progression and to examine the relationship between Mena[superscript INV] expression and markers for epithelial or mesenchymal status, stemness, stromal cell types and hypoxic regions. Further, while Mena[superscript INV] robustly expressed in vascularized areas of the tumor, it is not confined to cells adjacent to blood vessels. Altogether, these data demonstrate the specificity and utility of the anti-Mena[superscript INV]-isoform specific antibody, and provide the first description of endogenous Mena[superscript INV]protein expression in mouse and human tumors.United States. Dept. of Defense. Breast Cancer Research Program (Grants W81XWH-10-1-0040 and W81XWH-13-1-0031)National Institutes of Health (U.S.) (Grants U54-CA112967 and GM58801)Massachusetts Institute of Technology. Ludwig Center for Molecular Oncolog

Foxa2 and Cdx2 cooperate with Nkx2-1 to inhibit lung adenocarcinoma metastasis

Despite the fact that the majority of lung cancer deaths are due to metastasis, the molecular mechanisms driving metastatic progression are poorly understood. Here, we present evidence that loss of Foxa2 and Cdx2 synergizes with loss of Nkx2-1 to fully activate the metastatic program. These three lineage-specific transcription factors are consistently down-regulated in metastatic cells compared with nonmetastatic cells. Knockdown of these three factors acts synergistically and is sufficient to promote the metastatic potential of nonmetastatic cells to that of naturally arising metastatic cells in vivo. Furthermore, silencing of these three transcription factors is sufficient to account for a significant fraction of the gene expression differences between the nonmetastatic and metastatic states in lung adenocarcinoma, including up-regulated expression of the invadopodia component Tks5[subscript long], the embryonal proto-oncogene Hmga2, and the epithelial-to-mesenchymal mediator Snail. Finally, analyses of tumors from a genetically engineered mouse model and patients show that low expression of Nkx2-1, Foxa2, and Cdx2 strongly correlates with more advanced tumors and worse survival. Our findings reveal that a large part of the complex transcriptional network in metastasis can be controlled by a small number of regulatory nodes that function redundantly, and loss of multiple nodes is required to fully activate the metastatic program.National Cancer Institute (U.S.) (Cancer Center Support Grant P30-CA14051)Howard Hughes Medical InstituteNational Institutes of Health (U.S.) (Grant 5-U01-CA84306)United States. Dept. of Defense. Breast Cancer Research Program (U.S.) (Grant W81XWH-12-1-0031)Ludwig Center for Molecular Oncolog

PTP1B-dependent regulation of receptor tyrosine kinase signaling by the actin-binding protein Mena

During breast cancer progression, alternative mRNA splicing produces functionally distinct isoforms of Mena, an actin regulator with roles in cell migration and metastasis. Aggressive tumor cell subpopulations express Mena[superscript INV], which promotes tumor cell invasion by potentiating EGF responses. However, the mechanism by which this occurs is unknown. Here we report that Mena associates constitutively with the tyrosine phosphatase PTP1B and mediates a novel negative feedback mechanism that attenuates receptor tyrosine kinase signaling. On EGF stimulation, complexes containing Mena and PTP1B are recruited to the EGFR, causing receptor dephosphorylation and leading to decreased motility responses. Mena also interacts with the 5′ inositol phosphatase SHIP2, which is important for the recruitment of the Mena-PTP1B complex to the EGFR. When Mena[superscript INV] is expressed, PTP1B recruitment to the EGFR is impaired, providing a mechanism for growth factor sensitization to EGF, as well as HGF and IGF, and increased resistance to EGFR and Met inhibitors in signaling and motility assays. In sum, we demonstrate that Mena plays an important role in regulating growth factor–induced signaling. Disruption of this attenuation by Mena[superscript INV] sensitizes tumor cells to low–growth factor concentrations, thereby increasing the migration and invasion responses that contribute to aggressive, malignant cell phenotypes.Breast Cancer Research Program (U.S.) (Grant W81XWH-10-1-0040)Breast Cancer Research Program (U.S.) (Grant W81XWH-13-1-0031)National Institutes of Health (U.S.) (Grant U54-CA112967)National Institutes of Health (U.S.) (Grant GM58801)Virginia and D.K. Ludwig Fund for Cancer Researc

Tumor cell-driven extracellular matrix remodeling enables haptotaxis during metastatic progression

Fibronectin (FN) is a major component of the tumor microenvironment, but its role in promoting metastasis is incompletely understood. Here we show that FN gradients elicit directional movement of breast cancer cells, in vitro and in vivo. Haptotaxis on FN gradients requires direct interaction between α5β1 integrin and Mena, an actin regulator, and involves increases in focal complex signaling and tumor-cell-mediated extracellular matrix (ECM) remodeling. Compared to Mena, higher levels of the pro-metastatic MenaINV isoform associate with α5, which enables 3D haptotaxis of tumor cells towards the high FN concentrations typically present in perivascular space and in the periphery of breast tumor tissue. MenaINV and FN levels were correlated in two breast cancer cohorts, and high levels of MenaINV were significantly associated with increased tumor recurrence as well as decreased patient survival. Our results identify a novel tumor-cell-intrinsic mechanism that promotes metastasis through ECM remodeling and ECM guided directional migration.Ludwig Center for Molecular OncologyBreast Cancer Research Program (U.S.) (post-doctoral fellowship, W81-XWH-12-1-0031)National Institutes of Health (U.S.) (NIH grant U54-CA112967)National Institutes of Health (U.S.) (NIH grant R01 CA142833)Howard Hughes Medical InstituteNational Institutes of Health (U.S.) (NIH grant U01 CA143069)Kathy and Curt Marble Cancer Research Fund (Koch Institute Frontier Award)Prostate Cancer FoundationDavid H. Koch Institute for Integrative Cancer Research at MIT (NCI core grant P30-CA14051