Metastasis Suppressor Gene Inactivates Actin-Based Mechanism of Tumor Cell Motility
Preventing actin disassembly to decrease cell motility and metastasis: The actin filaments in this schematic representation of a polarized fibroblast are organized in several structures: lamellipodium, with projecting filopodium at the leading edge; stress fibers in the cell body and at the cell edge; and a loose actin network throughout the cell. Gelsolin binds and depolymerizes these actin filaments, allowing the cell to change its shape and move forward. A protein derived from the metastasis suppressor gene Nm23 binds and inactivates Gelsolin, slowing cell motility and preventing metastasis.
Metastasis is responsible for up to 90 percent of all cancer-related deaths. Though proteins derived from nearly a dozen metastasis suppressor genes have been discovered over the past 15 years, strategies for exploiting the proteins in metastasis-prevention therapies has been hampered by the lack of knowledge regarding the mechanisms underlying the proteins’ interactions with other proteins.
Patricia Steeg, Ph.D., Head of the Women’s Cancers Section in CCR’s Laboratory of Molecular Pharmacology, discovered the first metastasis suppressor gene, Nm23, in 1988. Nm23 has since been shown to profoundly suppress cell motility—important because cell motility underlies the ability of tumor cells to mobilize during metastasis. Recently, to better understand Nm23’s affect on cell motility, Steeg, Postdoctoral Fellow Natascia Marino, Ph.D., and their colleagues set out to identify protein-binding partners for Nm23-H1, a protein transcribed from the first of 10 known Nm23 genes.
Using the murine 4T1 breast carcinoma cell line, the researchers discovered a handful of Nm23-H1 binding partners employing a method called co-immunoprecipitation, in which an antibody is used to pull out of a lysate solution a known protein (such as Nm23-H1) that may be bound to other unknown proteins that can later be identified with mass spectrometry.
Among the candidate binding partners the researchers found, including a handful related to cell motility, was the actin-binding protein Gelsolin. The Nm23-H1-Gelsolin complex was verified with coimmunprecipitation assays in three other cellular models: the human breast carcinoma cell lines MDA-MB-23IT and MCF7, as well as the human melanoma cell line MDA-MB-435. Coimmunofluorescence revealed that Nm23-H1 and Gelsolin were colocalized to the cytoplasmic compartments of these cells.
Gelsolin facilitates the disassembly of actin, which is a critical step in cell motility. When Nm23-H1 was overexpressed, a fluorescence-based assay indicated that actin disassembly decreased, likely reducing the cell’s capacity for mobilization. The researchers discovered that overexpression of Gelsolin enhanced cell migration in 4T1 cells by 58 percent compared to a control vector, whereas overexpression of Nm23-H1 reduced cell migration by 46 percent. Similar effects were observed in the MDA-MB-231T and MCF7 cell lines.
In vivo overexpression of Gelsolin in 4T1 mice also led to increased tumor growth, which was reversed when Nm23-H1 was co-overexpressed. The researchers then examined two types of metastasis: discreet metastatic lesions and diffuse metastatic disease in the liver. Gelsolin overexpression in vivo increased discreet lesions by 24 percent. In addition, diffuse metastases were observed in 60 percent of mice, compared to none in control mice. Nm23-H1 overexpression reduced distinct lesions by 48 percent and no mice had diffuse metastases. Results were similar in the lungs of 4T1 mice.
In summary, Steeg and her team have identified a new mechanism by which Nm23 suppresses metastasis and tumor-cell motility. Across a variety of cell lines and cancer types, overexpression of the protein Nm23-H1 binds to Gelsolin and inhibits that protein’s ability to disassemble actin, greatly reducing cells’ capacity for mobilization.Summary Posted: Sun, 09/01/2013
Marino N, Marshall J-C, Collins JW, Zhou M, Qian Y, Veenstra T, Steeg PS. Nm23-H1 Binds to Gelsolin and Inactivates Its Actin-Severing Capacity to Promote Tumor Cell Motility and Metastasis. Cancer Research. August 12, 2013 PubMed Link