Activation of tissue plasminogen activator by metastasis-inducing S100P protein.

S100P protein in human breast cancer cells is associated with reduced patient survival and, in a model system of metastasis, it confers a metastatic phenotype upon benign mammary tumour cells. S100P protein possesses a C-terminal lysine residue. Using a multiwell in vitro assay, S100P is now shown for the first time to exhibit a strong, C-terminal lysine-dependent activation of tissue plasminogen activator (tPA), but not of urokinase-catalysed plasminogen activation. The presence of 10 µM calcium ions stimulates tPA activation of plasminogen 2-fold in an S100P-dependent manner. S100P physically interacts with both plasminogen and tPA in vitro, but not with urokinase. Cells constitutively expressing S100P exhibit detectable S100P protein on the cell surface, and S100P-containing cells show enhanced activation of plasminogen compared with S100P-negative control cells. S100P shows C-terminal lysine-dependent enhancement of cell invasion. An S100P antibody, when added to the culture medium, reduced the rate of invasion of wild-type S100P-expressing cells, but not of cells expressing mutant S100P proteins lacking the C-terminal lysine, suggesting that S100P functions outside the cell. The protease inhibitors, aprotinin or α-2-antiplasmin, reduced the invasion of S100P-expressing cells, but not of S100P-negative control cells, nor cells expressing S100P protein lacking the C-terminal lysine. It is proposed that activation of tPA via the C-terminal lysine of S100P contributes to the enhancement of cell invasion by S100P and thus potentially to its metastasis-promoting activity.

S100P dissociates myosin IIA filaments and focal adhesion sites to reduce cell adhesion and enhance cell migration.

S100 proteins promote cancer cell migration and metastasis. To investigate their roles in the process of migration we have constructed inducible systems for S100P in rat mammary and human HeLa cells that show a linear relationship between its intracellular levels and cell migration. S100P, like S100A4, differentially interacts with the isoforms of nonmuscle myosin II (NMIIA, K(d) = 0.5 µM; IIB, K(d) = 8 µM; IIC, K(d) = 1.0 µM). Accordingly, S100P dissociates NMIIA and IIC filaments but not IIB in vitro. NMIIA knockdown increases migration in non-induced cells and there is no further increase upon induction of S100P, whereas NMIIB knockdown reduces cell migration whether or not S100P is induced. NMIIC knockdown does not affect S100P-enhanced cell migration. Further study shows that NMIIA physically interacts with S100P in living cells. In the cytoplasm, S100P occurs in discrete nodules along NMIIA-containing filaments. Induction of S100P causes more peripheral distribution of NMIIA filaments. This change is paralleled by a significant drop in vinculin-containing, actin-terminating focal adhesion sites (FAS) per cell. The induction of S100P, consequently, causes significant reduction in cellular adhesion. Addition of a focal adhesion kinase (FAK) inhibitor reduces disassembly of FAS and thereby suppresses S100P-enhanced cell migration. In conclusion, this work has demonstrated a mechanism whereby the S100P-induced dissociation of NMIIA filaments leads to a weakening of FAS, reduced cell adhesion, and enhanced cell migration, the first major step in the metastatic cascade.

Targeted Destruction of S100A4 Inhibits Metastasis of Triple Negative Breast Cancer Cells.

Most patients who die of cancer do so from its metastasis to other organs. The calcium-binding protein S100A4 can induce cell migration/invasion and metastasis in experimental animals and is overexpressed in most human metastatic cancers. Here, we report that a novel inhibitor of S100A4 can specifically block its increase in cell migration in rat (IC50, 46 µM) and human (56 µM) triple negative breast cancer (TNBC) cells without affecting Western-blotted levels of S100A4. The moderately-weak S100A4-inhibitory compound, US-10113 has been chemically attached to thalidomide to stimulate the proteasomal machinery of a cell. This proteolysis targeting chimera (PROTAC) RGC specifically eliminates S100A4 in the rat (IC50, 8 nM) and human TNBC (IC50, 3.2 nM) cell lines with a near 20,000-fold increase in efficiency over US-10113 at inhibiting cell migration (IC50, 1.6 nM and 3.5 nM, respectively). Knockdown of S100A4 in human TNBC cells abolishes this effect. When PROTAC RGC is injected with mouse TNBC cells into syngeneic Balb/c mice, the incidence of experimental lung metastases or local primary tumour invasion and spontaneous lung metastasis is reduced in the 10-100 nM concentration range (Fisher's Exact test, p ≤ 0.024). In conclusion, we have established proof of principle that destructive targeting of S100A4 provides the first realistic chemotherapeutic approach to selectively inhibiting metastasis.

Self-association of calcium-binding protein S100A4 and metastasis.

Elevated levels of the calcium-binding protein S100A4 promote metastasis and in carcinoma cells are associated with reduced survival of cancer patients. S100A4 interacts with target proteins that affect a number of activities associated with metastatic cells. However, it is not known how many of these interactions are required for S100A4-promoted metastasis, thus hampering the design of specific inhibitors of S100A4-induced metastasis. Intracellular S100A4 exists as a homodimer through previously identified, well conserved, predominantly hydrophobic key contacts between the subunits. Here it is shown that mutating just one key residue, phenylalanine 72, to alanine is sufficient to reduce the metastasis-promoting activity of S100A4 to 50% that of the wild type protein, and just 2 or 3 specific mutations reduces the metastasis-promoting activity of S100A4 to less than 20% that of the wild type protein. These mutations inhibit the self-association of S100A4 in vivo and reduce markedly the affinity of S100A4 for at least two of its protein targets, a recombinant fragment of non-muscle myosin heavy chain isoform A, and p53. Inhibition of the self-association of S100 proteins might be a novel means of inhibiting their metastasis-promoting activities.

The Role of the C-Terminal Lysine of S100P in S100P-Induced Cell Migration and Metastasis.

S100P protein is a potent inducer of metastasis in a model system, and its presence in cancer cells of patients is strongly associated with their reduced survival times. A well-established Furth Wistar rat metastasis model system, methods for measuring cell migration, and specific inhibitors were used to study pathways of motility-driven metastasis. Cells expressing C-terminal mutant S100P proteins display markedly-reduced S100P-driven metastasis in vivo and cell migration in vitro. These cells fail to display the low focal adhesion numbers observed in cells expressing wild-type S100P, and the mutant S100P proteins exhibit reduced biochemical interaction with non-muscle myosin heavy chain isoform IIA in vitro. Extracellular inhibitors of the S100P-dependent plasminogen activation pathway reduce, but only in part, wild-type S100P-dependent cell migration; they are without effect on S100P-negative cells or cells expressing C-terminal mutant S100P proteins and have no effect on the numbers of focal adhesions. Recombinant wild-type S100P protein, added extracellularly to S100P-negative cells, stimulates cell migration, which is abolished by these inhibitors. The results identify at least two S100P-dependent pathways of migration, one cell surface and the other intracellularly-linked, and identify its C-terminal lysine as a target for inhibiting multiple migration-promoting activities of S100P protein and S100P-driven metastasis.

The basic C-terminal amino acids of calcium-binding protein S100A4 promote metastasis.

The calcium-binding protein S100A4 can induce a metastatic phenotype in animal model systems and its expression in various human cancers has been shown to be associated with metastasis and reduced patient survival. Using a series of nested deletion mutants, it is now shown that the two C-terminal lysine residues are required for the enhanced metastasis, invasion and migration abilities that S100A4 confers on cells in a model system of metastasis. Basic C-terminal residues enhance the affinity between S100A4 and its best characterized target, a recombinant C-terminal fragment of non-muscle myosin II heavy chain isoform A (NMMHC-IIA). In wild-type S100A4 protein, the presence of the C-terminal lysine, residue 101, enhances the rate of association between S100A4 and NMMHC-IIA. These results identify the amino acids of S100A4 that are involved in metastasis induction and show that the C-terminal region of S100A4 is a possible target for inhibitors of its metastatic action.

S100P enhances the motility and invasion of human trophoblast cell lines.

S100P has been shown to be a marker for carcinogenesis where its expression in solid tumours correlates with metastasis and a poor patient prognosis. This protein's role in any physiological process is, however, unknown. Here we first show that S100P is expressed both in trophoblasts in vivo as well as in some corresponding cell lines in culture. We demonstrate that S100P is predominantly expressed during the early stage of placental formation with its highest expression levels occurring during the first trimester of gestation, particularly in the invading columns and anchoring villi. Using gain or loss of function studies through overexpression or knockdown of S100P expression respectively, our work shows that S100P stimulates both cell motility and cellular invasion in different trophoblastic and first trimester EVT cell lines. Interestingly, cell invasion was seen to be more dramatically affected than cell migration. Our results suggest that S100P may be acting as an important regulator of trophoblast invasion during placentation. This finding sheds new light on a hitherto uncharacterized molecular mechanism which may, in turn, lead to the identification of novel targets that may explain why significant numbers of confirmed human pregnancies suffer complications through poor placental implantation.

S100A4 Elevation Empowers Expression of Metastasis Effector Molecules in Human Breast Cancer.

Many human glandular cancers metastasize along nerve tracts, but the mechanisms involved are generally poorly understood. The calcium-binding protein S100A4 is expressed at elevated levels in human cancers, where it has been linked to increased invasion and metastasis. Here we report genetic studies in a Drosophila model to define S100A4 effector functions that mediate metastatic dissemination of mutant Ras-induced tumors in the developing nervous system. In flies overexpressing mutant RasVal12 and S100A4, there was a significant increase in activation of the stress kinase JNK and production of the matrix metalloproteinase MMP1. Genetic or chemical blockades of JNK and MMP1 suppressed metastatic dissemination associated with S100A4 elevation, defining required signaling pathway(s) for S100A4 in this setting. In clinical specimens of human breast cancer, elevated levels of the mammalian paralogs MMP2, MMP9, and MMP13 are associated with a 4- to 9-fold relative decrease in patient survival. In individual tumors, levels of MMP2 and MMP13 correlated more closely with levels of S100A4, whereas MMP9 levels correlated more closely with the S100 family member S100P. Overall, our results suggest the existence of evolutionarily conserved pathways used by S100A4 to promote metastatic dissemination, with potential prognostic and therapeutic implications for metastasis by cancers that preferentially exploit nerve tract migration routes. Cancer Res; 77(3); 780-9. ©2016 AACR.

Regulation of dinucleoside polyphosphate pools by the YgdP and ApaH hydrolases is essential for the ability of Salmonella enterica serovar typhimurium to invade cultured mammalian cells.

The ygdP and apaH genes of Salmonella enterica serovar Typhimurium (S. Typhimurium) encode two unrelated dinucleoside polyphosphate (NpnN) hydrolases. For example, YgdP cleaves diadenosine tetraphosphate (Ap4A) producing AMP and ATP, while ApaH cleaves Ap4A producing 2ADP. Disruption of ygdP, apaH individually, and disruption of both genes together reduced intracellular invasion of human HEp-2 epithelial cells by S. Typhimurium by 9-, 250-, and 3000-fold, respectively. Adhesion of the mutants was also greatly reduced compared with the wild type. Invasive capacity of both single mutants was restored by transcomplementation with the ygdP gene, suggesting that loss of invasion was due to increased intracellular NpnN. The normal level of 3 microM adenylated NpnN (ApnN) was increased 1.5-, 3.5-, and 10-fold in the ygdP, apaH and double mutants, respectively. Expression of the putative ptsP virulence gene downstream of ygdP was not affected in the ygdP mutant. Analysis of 19 metabolic enzyme activities and the ability to use a range of carbohydrate carbon sources revealed a number of differences between the mutants and wild type. The increase in intracellular NpnN in the mutants appears to cause changes in gene expression that limit the ability of S. Typhimurium to adhere to and invade mammalian cells.