The major problem with cancer is the ability of cancer cells to infiltrate surrounding tissue (invasion) or to spread to distant organs (metastasis), thereby decreasing the patient’s survival. One of the first steps in the process of metastasis is local spreading or invasion from the primary tumour and certain Ca2+ -binding proteins of the S100 family eg, S100A4 and S100P, can induce a metastatic phenotype and enhance cell migration and invasion. Both S100A4 and S100P are expressed at elevated levels in several forms of cancer including breast, pancreatic, prostate, colon, ovary, oesophagus and lung cancer. Initially, we introduced a series of deletion mutants on the S100A4 protein by truncation of the C-terminal region. The S100A4 open-reading-frame cloned into an expression vector was subjected to site-directed mutagenesis to create the mutations, either by deletion or by substitution. These mutant proteins, when expressed in cells, conferred reduced metastasis, invasion and migration-inducing abilities, compared to cells expressing wild type S100A4 protein. The mutations reduced the ability of the S100A4 protein to bind to one of its intracellular targets, non-muscle myosin II heavy chain isoform A (NMMHCIIA) in vitro. There was a significant reduction in metastasis, migration and binding to NMMHCIIA when only the last two basic C-terminal lysine residues (Lys100, 101) were removed. A similar decrease in metastasis, migration, and invasion was also observed in mutated S100P protein. Removing the single C-terminal lysine residue from S100P protein, or its replacement by alanine, dramatically reduced the metastasis-promoting ability of this protein. Binding to NMMHCIIA was also tested in vitro. The results showed that S100P mutant protein exhibited 10-folds lower affinity to NMMHCIIA than to the wild type protein. In vivo, cells expressing high levels of S100P mutant protein exhibited a dramatically changed organisation of the NMMHCIIA cytoskeleton compared to cells expressing wild type S100P. There was also change in the actin cytoskeleton to a more polarised, highly bundled structure in cells with an elevated level of mutant S100P protein compared to cells expressing the wild type protein. These results identify the lysine(s) at the C-terminal of these two S100 family proteins as being important in their binding to one of their target proteins, in cell migration, cytoskeletal organisation, cell invasion and metastasis. These observations may have pharmaceutical implications for potential anti-cancer drugs targeted to S100A4 and S100P. This work was supported by the James Tudor Foundation and the Cancer and Polio Research Fund.

Thamir Ismail, Stephane Gross, Connie Goh, Mark C. Wilkinson, Philip S. Rudland and Roger Barraclough .


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  • Received: 16 May 2012
  • Accepted: 16 May 2012
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