MenaINV Expression Research Articles

Abstract C41: MenaINV dysregulation of receptor tyrosine kinase signaling

Abstract Mena, an actin regulatory protein, is upregulated in human breast cancer and its expression is associated with increased metastatic progression. An invasion-specific isoform, MenaINV, is upregulated selectively in invasive tumor cells and increases invasion and metastasis by potentiating tumor cell responses to EGF. MenaINV causes dysregulation of the tyrosine phosphatase PTP1B, leading to increased phosphorylation of EGFR and other downstream targets. We investigated whether MenaINV could similarly promote invasion through dysregulation of signaling downstream of other receptor tyrosine kinases (RTKs). The behavior of MDA-MB-231 triple negative breast cancer cells stably expressing MenaINV or the other cannonical, broadly expressed Mena lacking the “INV” exon was measured after growth factor treatment in two migration assays: a 2D protrusion assay on matrigel and collagen coated coverslips and a 3D invasion assay into collagen gels. We found that expression of MenaINV sensitized responses to other EGFR ligands including HB-EGF and amphiregulin. In addition, MenaINV expression enhanced responses mediated by two other RTKs associated with tumor progression, Met and IGFR, both known PTP1B substrates. Cells expressing MenaINV respond to 20-fold lower concentrations HGF and IGF than control cells. However, there was no effect of MenaINV on responses to the ERBB3 ligand neuregulin and the chemokine SDF-1. Recently we reported that Mena can regulate cell motility in fibroblasts via its interaction with α5β1 integrin, which is known to co-traffic with EGFR and other RTKs to regulate invasion in tumor cells expressing mutant p53 or upon inhibition of αvβ3. We asked whether the direct interaction of Mena with α5β1 also contributes to invasion. Disrupting the interaction between the Mena isoforms and α5β1 in MDA-MB-231 cells affected how cells responded to EGF in a 3D collagen gel. In addition, fibroblasts that were null for Mena were unable to respond to a fibronectin gradient in a haptotaxis assay. Overall, these results suggest that the effects of MenaINV on invasion and metastasis could be mediated via different RTKs, as well as through direct interaction with integrins and the extracellular matrix. Experiments are currently underway to investigate how MenaINV regulates signalling downstream of these RTKs and integrins, to understand the molecular mechanism underlying the pro-metastatic effect of MenaINV. Citation Format: Madeleine J. Oudin, Shannon K. Hughes-Alford, Miles Miller, Sreeja B. Asokan, James E. Bear, Doug A. Lauffenburger, Frank B. Gertler. MenaINV dysregulation of receptor tyrosine kinase signaling. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C41.

MenaINV Isoform Expression in Human Invasive Ductal Carcinomas of the Breast Correlates with Tumor Microenvironment of Metastatsis (TMEM) Score; a Pilot Study Indicating the Potential Use of MenaINV/11a Ratio as a Predictor of Distant Metastases.

Abstract Background: Primary mammary carcinomas in rodents contain a subpopulation of invasive tumor cells that move towards perivascular macrophages and intravasate. An isoform of actin regulatory protein Mena, MenaINV is up-regulated in these invasive cells, while isoform Mena 11a is down regulated (Goswami et al 2009). The direct apposition of Mena positive tumor cells to endothelial cells and perivascular macrophages has been described as Tumor Microenvironment of Metastasis (TMEM) and its density is shown to be a predictor of metastatic outcome in human invasive ductal carcinomas of the breast (Robinson et al 2009). We questioned if MenaINV and Mena 11a isoform expression correlates with the invasiveness of breast lesions in Polyoma Middle T antigen (PyMT) mouse mammary tumors and human invasive ductal carcinomas (IDC), as well as if increased MenaINV expression is associated with high TMEM counts in human IDCs.Materials and Methods: We analyzed MenaINV and Mena 11a expression in fine needle aspiration biopsy (FNA) samples from 18 mice with PyMT mammary tumors, 3 human IDCs and 1 fibroadenoma (FA) by qRT-PCR. The expression of Mena isoforms from in situ PyMT lesions was compared with metastatic PyMT tumors and expressed as fold change. Mena isoform levels from IDC were compared to that in FA and expressed as fold change. Representative sections from IDCs were triple immunostained with anti-Mena, anti-CD 68 and anti-CD34 and independently analyzed for TMEM counts on ten x400 fields by two pathologists.Results: We observed a 7.5 fold increase in MenaINV expression and a 3 fold decrease of Mena 11a in metastatic PyMT tumors when compared to in situ lesions. The results from human samples are shown in Figure 1. The top panel shows MenaINV and Mena 11a fold change in IDCs compared to FA. The bottom panel shows TMEM counts for each IDC and MenaINV/11a ratios. Both, MenaINV expression and TMEM count varied significantly, whereas Mena 11a was exceedingly low in IDCs. In all 3 IDCs the levels of MenaINV correlated with TMEM counts.Discussion: This pilot study indicates for the first time that the expression of MenaINV and Mena 11a can be successfully analyzed in FNA biopsy samples from human breast lesions. In combination with the TMEM count, Mena isoforms can potentially become useful markers for predicting the metastatic potential of an individual breast lesion. A larger study is underway to develop a scoring system combining Mena isoform expression with the TMEM count.Figure 1: Top panel: qRT-PCR assessment of Mena splice variant expression in FNA samples from human breast lesions. Levels of MenaINV and Mena 11a in 3 IDCs were compared to those in FA. The result is expressed as fold change. Bottom panel: TMEMs on tissue sections from IDC with TMEM counts and MenaINV/11a ratios. Circles represent TMEMs. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2140.