Poster Sessions

CANCER-35

Jean-Claude Marshall

J.C. Marshall, J. Collins, Z. Ming, T. Veenstra, C. Khanna, P. Steeg

An Unbiased Screen for Nm23-H1 Interacting Proteins Identifies the Cytoskeletal Regulator Ezrin

Nm23-H1 was the first in a novel class of genes to be characterized as inhibiting the formation of metastasis while leaving the growth of the primary tumor unchanged. An unbiased survey of Nm23-H1 binding proteins was conducted in a 4T1 murine mammary carcinoma model system. Flag tagged Nm23-H1 and its mouse variant, -M1, were transfected into 4T1 mouse mammary cancer cells labeled with luciferase. Clonal lines were inoculated into the number four mammary fat pad of mice and allowed to grow for 10 days prior to excision due to size of the primary tumor. Primary tumor size was unaffected by overexpression of Nm23-H1 or –M1. Histologic examination revealed a 75% decrease in liver metastasis by Nm23-H1 or –M1 overexpression (P = 0.001). Flag tagged Nm23 from both H1 and M1 clones were immunoprecipitated (IP) from in vitro cell lines as well as isolated primary tumors, lymph nodes, spleen and liver tissue after verification of metastasis by histological examination of the tissue. Mass spectrometry analysis of IP samples from cell lines and primary tumors showed 99 proteins of interest. From these, 9 previously described Nm23 interacting proteins were. Co-immunoprecipitation of Ezrin and Nm23-H1 has been confirmed using both Nm23 and Ezrin as the immunoprecipitating antibodies. Ezrin belongs to a family of widely expressed proteins known as ERMs which have been described to link the actin cytoskeleton to membrane and membrane-associated proteins. Colocalization experiments using confocal imaging, have shown a potential dynamic interaction between Nm23 and Ezrin, where Ezrin becoming delocalized from Nm23 when tumor cells become motile.