The Role of MenaINV In Promoting Tumor Cell Invadopodium Maturation

Abstract

Breast cancer, the most common form of cancer in women, is highly lethal due to its propensity for dissemination from the primary tumor and metastasis to distant sites. Yet there has been relatively little success in developing treatments to prevent metastasis. A better understanding of the changes in gene expression and protein-protein interaction required for the transformation of a naive to invasive tumor cell population would allow one to more accurately assess a patient's risk of malignant disease and therapeutically target bottlenecks in the progression to metastasis.;An "invasion signature" has been previously developed by comparing gene expression changes in invasive breast carcinoma cells relative to the whole primary tumor. Prominent within this invasion signature are proteins that influence the formation of actinbased protrusions that facilitate invasion, termed invadopodia. The actin-binding protein Mena was found to be highly upregulated in the invasive cells, and a splice-isoform of Mena not usually expressed in epithelial cells (MenaINV) was particularly upregulated, while an isoform associated with premalignant breast tissue (Mena11a) was downregulated. It has since been shown that MenaINV expression enhances (a) tumor cell migration, (b) tumor cell invasion through dense extracellular barriers, (c) penetration of tumor cells into blood vessels (intravasation), and (d) metastasis of breast tumor cells to distal organs. However, the mechanisms that enable MenaINV to alter this variety of tumor cell phenotypes, contributing to metastasis, are poorly understood.;Initial investigation into such mechanisms has shown that MenaINV prevents the normal recruitment of the phosphatase PTP1B to the EGF receptor, and that the resulting increase in EGF signaling by MenaINV enhanced leading edge protrusion and locomotion of tumor cells. Yet these results did not explain findings showing that MenaINV was able to enhance proteolysis-dependent tumor invasion, and specifically that MenaINV increased invadopodium-based matrix degradation. Since several steps of the metastatic cascade rely on focal degradation of extracellular barriers to tumor migration, and MenaINV has been shown to increase invadopodium-based matrix degradation, I focused on understanding which parts of invadopodium formation and function were influenced by MenaINV relative to the ubiquitously expressed classic isoform, Mena, and Mena11a. To test my initial hypothesis that MenaINV specifically promotes invadopodium maturation, Mena, MenaINV or Mena11a were independently overexpressed in breast tumor cell lines. MenaINV produced a substantially larger increase in the number of mature invadopodia than Mena, which produced a smaller increase only in cell lines expressing endogenous MenaINV. In contrast, Mena11a overexpression had no effect at all. Importantly, by knocking down MenaINV, I found that the level of invadopodium maturation depends specifically on MenaINV, confirming my hypothesis.;Next, I demonstrated that MenaINV localizes to invadopodia following the assembly of the precursor core complex, but prior to invadopodium stabilization and protrusion required for subsequent maturation. In addition, I provided evidence that MenaINV localizes to nearly all mature invadopodia, but less than half of invadopodium precursors, suggesting that MenaINV has a continuing function at mature invadopodia that is consistent with MenaINV-induced increases in invadopodium lifetime. Then, I investigated the functional role of MenaINV during invadopodium maturation. Since it was previously known that cortactin phosphorylation at tyrosine 421 (Y421) is required for invadopodium maturation, I theorized that MenaINV could be involved in this process. I demonstrated that MenaINV indeed increases the basal phosphorylation level at Y421 in unstimulated cells. Furthermore, PTP1B overexpression decreased cortactin phosphorylation at Y421 and inhibited invadopodium maturation, while PTP1B knock down increased phosphorylation at Y421 in invadopodia and promoted invadopodium maturation. Finally, I determined that MenaINV localization to invadopodium precursors reduces the level of PTP1B localizing to those precursors.;Taken together, my work demonstrates that MenaINV has a unique role amongst the Mena isoforms in promoting invadopodium maturation. Mena INV arrives during invadopodium precursor stabilization and persists at mature invadopodia, where it prevents PTP1B localization. Reduced levels of PTP1B at the invadopodium precursor promote cortactin phosphorylation at Y421, which is required for actin polymerization at invadopodia and invadopodium maturation. Thus, MenaINV promotes invadopodium maturation through cortactin phosphorylation, a downstream effector of extracellular signals known to promote invadopodium maturation such as EGF and â1-integrin. My work suggests that MenaINV promotes invasion and metastasis by enabling tumor cells to efficiently produce stable and proteolytically active invadopodia in response to stimuli in a wide range of microenvironments. My results also help explain the success of Mena isoform expression patterns as prognostics in assessing the risk of distant metastasis.