Inhibition of the invasion and metastasis of mammary carcinoma cells by NBD peptide targeting S100A4 via the suppression of the Sp1/MMP­14 axis.

A synthetic peptide that blocks the interaction between the metastasis­enhancing calcium­binding protein, S100A4, and its effector protein, methionine aminopeptidase 2 (MetAP2) (the NBD peptide), was previously demonstrated to inhibit the angiogenesis of endothelial cells, leading to the regression of human prostate cancer in a xenograft model. However, the effects of the NBD peptide on the malignant properties of cancer cells that express S100A4 remain to be elucidated. The present study demonstrates that the NBD peptide inhibits the invasiveness and metastasis of highly metastatic human mammary carcinoma cells. The introduction of the peptide into MDA­MB­231 variant cells resulted in the suppression of matrix degradation in a gelatin invadopodia assay and invasiveness in a Matrigel invasion assay. In line with these results, the peptide significantly downregulated the expression of matrix metalloproteinase (MMP)­14 (MT1­MMP). Mechanistic analysis of the downregulation of MMP­14 revealed the suppression of the expression of the transcription factor, specificity protein 1 (Sp1), but not that of nuclear factor (NF)­κB, early growth response 1 (EGR1) or ELK3, all of which were reported to be involved in transcriptional regulation of the MMP­14 gene. At the same time, evidence suggested that the NBD peptide also suppressed Sp1 and MMP­14 expression levels in MDA­MB­468 cells. Importantly, the intravenous administration of the NBD peptide encapsulated in liposomes inhibited pulmonary metastasis from mammary gland tumors in mice with xenograft tumors. These results indicate that the NBD peptide can suppress malignant tumor growth through the suppression of the Sp1/MMP­14 axis. Taken together, these results reveal that the NBD peptide acts on not only endothelial cells, but also on tumor cells in an integrated manner, suggesting that the peptide may prove to be a promising cancer therapeutic peptide drug.

Structural features of methionine aminopeptidase2-active core peptide essential for binding with S100A4.

Methionine aminopeptidase 2 (MetAP2) is one of the effector proteins of S100A4, a metastasis-associated calcium-binding protein. This interaction is involved in angiogenesis. The region of MetAP2 that interacts with S100A4 includes amino acids 170 to 208. A peptide corresponding to this region, named as NBD, has potent anti-angiogenic activity and suppresses tumor growth in a xenograft cancer model. However, the binding mode of NBD to S100A4 was totally unknown. Here we describe our analysis of the relationship between the inhibitory activity and the structure of NBD, which adopts a characteristic helix-turn-helix structure as shown by X-ray crystallographic analysis, and peptide fragments of NBD. We conducted physicochemical analyses of the interaction between S100A4 and the peptides, including surface plasmon resonance, microscale thermophoresis, and circular dichroism, and performed docking/molecular dynamics simulations. Active peptides had stable secondary structures, whereas inactive peptides had a little secondary structure. A computational analysis of the interaction mechanism led to the design of a peptide smaller than NBD, NBD-ΔN10, that possessed inhibitory activity. Our study provides a strategy for design for a specific peptide inhibitor against S100A4 that can be applied to the discovery of inhibitors of other protein-protein interactions.

Correlation of two distinct metastasis-associated proteins, MTA1 and S100A4, in angiogenesis for promoting tumor growth.

Extensive studies on metastasis-associated proteins, S100A4 and MTA1, have been carried out for over two decades, but correlation of both proteins remains obscure. Here we show evidence for the correlation in angiogenesis. First, silencing of each protein by siRNA-mediated knockdown in mouse endothelial MSS31 cells resulted in the inhibition of tube formation. Unexpectedly, the knockdown of MTA1 affected not only its own expression but also the expression of S100A4, whereas silencing of S100A4 did not affect the MTA1 expression. Additionally, non-muscle myosin IIA (NMIIA) phosphorylation, which was partly controlled by S100A4, was found to be upregulated by knockdown of both proteins in MSS31 cells. Moreover, cycloheximide treatment of MSS31 cells revealed that the rate of S100A4 degradation was accelerated by MTA1 knockdown. This finding, together with our observation that cytoplasmic MTA1, but not nuclear MTA1, was colocalized with S100A4, suggested the involvement of MTA1 in S100A4 stability. The direct in vivo angiogenesis assay showed that both protein siRNAs provoked a significant inhibition of new blood vessel formation induced by angiogenic factors, indicating their anti-angiogenic activities. Treatment of human pancreatic tumor (PANC-1) xenograft in mice with mMTA1 siRNA resulted in tumor regression via suppression of angiogenesis in vivo, as also observed in the case of human prostate cancer xenograft treated with mS100A4 siRNA. Taken together, these data led us to conclude that the MTA1-S100A4-NMIIA axis exists in endothelial cells as a novel pathway in promoting tumor vascular formation and could be a target for suppressing tumor growth and metastasis.

Efficient inhibition of tumor angiogenesis and growth by a synthetic peptide blocking S100A4-methionine aminopeptidase 2 interaction.

The prometastatic calcium-binding protein, S100A4, is expressed in endothelial cells, and its downregulation markedly suppresses tumor angiogenesis in a xenograft cancer model. Given that endothelial S100A4 can be a molecular target for inhibiting tumor angiogenesis, we addressed here whether synthetic peptide capable of blocking S100A4-effector protein interaction could be a novel antiangiogenic agent. To examine this hypothesis, we focused on the S100A4-binding domain of methionine aminopeptidase 2, an effector protein, which plays a role in endothelial cell growth. Overexpression of the domain in mouse endothelial MSS31 cells reduced DNA synthesis, and the corresponding synthetic peptide (named NBD) indeed interacted with S100A4 and inhibited capillary formation in vitro and new blood vessel formation in vivo. Intriguingly, a single intra-tumor administration of the NBD peptide in human prostate cancer xenografts significantly reduced vascularity, resulting in tumor regression. Mechanistically, the NBD peptide enhanced assembly of nonmuscle myosin IIA filaments along with Ser1943 phosphorylation, stimulated formation of focal adhesions without phosphorylation of focal adhesion kinase, and provoked G1/S arrest of the cell cycle. Altogether, the NBD peptide is a potent inhibitor for tumor angiogenesis, and is the first example of an anticancer peptide drug developed on the basis of an endothelial S100A4-targeted strategy.

Silencing of S100A4, a metastasis-associated protein, in endothelial cells inhibits tumor angiogenesis and growth.

Endothelial cells express S100A4, a metastasis-associated protein, but its role in angiogenesis remains to be elucidated. Here we show that knockdown of S100A4 in mouse endothelial MSS31 cells by murine specific small interference RNA (mS100A4 siRNA) markedly suppressed capillary-like tube formation in vitro, in early stage after the treatment, along with down- and up-regulation of some of the pro-angiogenic and anti-angiogenic gene expression, respectively. Of particular note is that intra-tumor administration of the mS100A4 siRNA in a human prostate cancer xenograft significantly reduced tumor vascularity and resulted in the inhibition of tumor growth. These findings show that S100A4 in endothelial cells is involved in tube formation, and suggest its potential as a molecular target for inhibiting tumor angiogenesis, which warrants further development of endothelial S100A4-based strategies for cancer treatment.

High expression of methionine aminopeptidase type 2 in germinal center B cells and their neoplastic counterparts.

Methionine aminopeptidase type 2 (MetAP2) is a bifunctional protein that plays critical roles in the regulation of protein synthesis and post-translational processing by (a) protecting the alpha subunit of eukaryotic initiation factor 2 from inhibitory phosphorylation by eukaryotic initiation factor 2 kinases and (b) removing the amino-terminal methionine residue from nascent protein. MetAP2 is also known as the molecular target of the angiogenesis inhibitor TNP-470. In addition, it has been recently suggested that MetAP2 has an antiapoptotic function in mesothelioma. To know the pattern of expression of MetAP2 in normal and neoplastic tissues, we raised two specific rabbit polyclonal Abs and examined the pattern of MetAP2 expression in various normal and pathologic specimens. Unexpectedly, we found a very high and selective expression of MetAP2 in germinal center B cells. In the germinal center, dark zone B cells tended to express more MetAP2 than light zone B cells. When 200 malignant lymphomas of various subtypes were studied, a high level of MetAP2 expression, equivalent to that observed in germinal center B cells, was noted exclusively on B-cell lymphoma subtypes that are currently regarded as the neoplastic counterparts of germinal center B cells. The expression of MetAP2 in diffuse large B-cell lymphomas correlated well with that of BCL6 (p < 0.05) but not with that of either CD10 or BCL2. These data suggest that MetAP2 has specific function(s) in germinal center B cells and that the function is shared by neoplastic counterparts of germinal center B cells.

Methionine aminopeptidase 2 is a new target for the metastasis-associated protein, S100A4.

S100A4 is an EF-hand type calcium-binding protein that regulates tumor metastasis and a variety of cellular processes via interaction with different target proteins. Here we report that S100A4 physically interacts with methionine aminopeptidase 2 (MetAP2), the primary target for potent angiogenesis inhibitors, fumagillin and ovalicin. Using a yeast two-hybrid screen, S100A4 was found to interact with the N-terminal half of MetAP2. In vitro pull-down assays showed that S100A4 associates with MetAP2 in a calcium-dependent manner. In addition, the binding site of S100A4 was found located within the region between amino acid residues 170 and 229 of MetAP2. In vivo interaction of S100A4 with MetAP2 was verified by co-immunoprecipitation analysis. Immunofluorescent staining revealed that S100A4 and MetAP2 were co-localized in both quiescent and basic fibroblast growth factor-treated murine endothelial MSS31 cells, in the latter of which a significant change of intracellular distribution of both proteins was observed. Although the binding of S100A4 did not affect the in vitro methionine aminopeptidase activity of MetAP2, the cytochemical observation suggests a possible involvement of S100A4 in the regulation of MetAP2 activity through changing its localization, thereby modulating the N-terminal methionine processing of nascent substrates. These results may offer an essential clue for understanding the functional role of S100A4 in regulating endothelial cell growth and tumor metastasis.