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The tumor-elicited inflammation is closely related to tumor microenvironment during tumor progression. S100A8, an endogenous ligand of Toll-like receptor 4 (TLR4), is known as a key molecule in the tumor microenvironment and premetastatic niche formation. We firstly generated a novel multivalent S100A8 competitive inhibitory peptide (divalent peptide3A5) against TLR4/MD-2, using the alanine scanning. Divalent peptide3A5 suppressed S100A8-mediated interleukin-8 and vascular endothelial growth factor production in human colorectal tumor SW480 cells. Using SW480-transplanted xenograft models, divalent peptide3A5 suppressed tumor progression in a dose-dependent manner. We demonstrated that combination therapy with divalent peptide3A5 and bevacizumab synergistically suppressed tumor growth in SW480 xenograft models. Using syngeneic mouse models, we found that divalent peptide3A5 improved the efficacy of anti-programmed death (PD)1 antibody, and lung metastasis. In addition, by using multivalent peptide library screening based on peptide3A5, we then isolated two more candidates; divalent ILVIK, and tetravalent ILVIK. Of note, multivalent ILVIK, but not monovalent ILVIK showed competitive inhibitory activity against TLR4/MD-2 complex, and anti-tumoral activity in SW480 xenograft models. As most tumor cells including SW480 cells also express TLR4, S100A8 inhibitory peptides would target both the tumor microenvironment and tumor cells. Thus, multivalent S100A8 inhibitory peptides would provide new pharmaceutical options for aggressive cancers.

The tumor-elicited inflammation is closely related to tumor microenvironment during tumor progression. S100A8, an endogenous ligand of Toll-like receptor 4 (TLR4), is known as a key molecule in the tumor microenvironment and premetastatic niche formation. We firstly generated a novel multivalent S100A8 competitive inhibitory peptide (divalent peptide3A5) against TLR4/MD-2, using the alanine scanning. Divalent peptide3A5 suppressed S100A8-mediated interleukin-8 and vascular endothelial growth factor production in human colorectal tumor SW480 cells. Using SW480-transplanted xenograft models, divalent peptide3A5 suppressed tumor progression in a dose-dependent manner. We demonstrated that combination therapy with divalent peptide3A5 and bevacizumab synergistically suppressed tumor growth in SW480 xenograft models. Using syngeneic mouse models, we found that divalent peptide3A5 improved the efficacy of anti-programmed death (PD)1 antibody, and lung metastasis. In addition, by using multivalent peptide library screening based on peptide3A5, we then isolated two more candidates; divalent ILVIK, and tetravalent ILVIK. Of note, multivalent ILVIK, but not monovalent ILVIK showed competitive inhibitory activity against TLR4/MD-2 complex, and anti-tumoral activity in SW480 xenograft models. As most tumor cells including SW480 cells also express TLR4, S100A8 inhibitory peptides would target both the tumor microenvironment and tumor cells. Thus, multivalent S100A8 inhibitory peptides would provide new pharmaceutical options for aggressive cancers.

Emerging evidence suggests that neural activity contributes to tumor initiation and its acquisition of metastatic properties. More specifically, it has been reported that the sympathetic nervous system regulates tumor angiogenesis, tumor growth, and metastasis. The function of the sympathetic nervous system in primary tumors has been gradually elucidated. However, its functions in pre-metastatic environments and/or the preparation of metastatic environments far from the primary sites are still unknown. To investigate the role of the sympathetic nervous system in pre-metastatic environments, we performed chemical sympathectomy using 6-OHDA in mice and observed a decrease in lung metastasis by attenuating the recruitment of myeloid-derived suppressor cells. Furthermore, we note that neuro-immune cell interactions could be observed in tumor-bearing mouse lungs in conjunction with the decreased expression of Sema3A. These data indicate that the sympathetic nervous system contributes to the preparation of pre-metastatic microenvironments in the lungs, which are mediated by neuro-immune cell interactions.

Accumulating evidence indicates that an elevated ephrin-A1 expression is positively correlated with a worse prognosis in some cancers such as colon and liver cancer. The detailed mechanism of an elevated ephrin-A1 expression in a worse prognosis still remains to be fully elucidated. We previously reported that ADAM12-cleaved ephrin-A1 enhanced lung vascular permeability and thereby induced lung metastasis. However, it is still unclear whether or not cleaved forms of ephrin-A1 are derived from primary tumors and have biological activities. We identified the ADAM12-mediated cleavage site of ephrin-A1 by a Matrix-assisted laser desorption ionization mass spectrometry and checked levels of ephrin-A1 in the serum and the urine derived from the primary tumors by using a mouse model. We found elevated levels of tumor-derived ephrin-A1 in the serum and the urine in the tumor-bearing mice. Moreover, inhibition of ADAM-mediated cleavage of ephrin-A1 or antagonization of the EphA receptors resulted in a significant reduction of lung metastasis. The results suggest that tumor-derived ephrin-A1 is not only a potential biomarker to predict lung metastasis from the primary tumor highly expressing ephrin-A1 but also a therapeutic target of lung metastasis.

The deregulation of Eph/ephrin protein expression has been shown to lead to tumor development and progression. Both mRNA and protein expression analyses using clinical samples have demonstrated that ephrin-A1 is over-expressed in various cancers and positively correlates with a poor prognosis for cancer patients. The prognosis of cancer patients depends on metastasis to distant organs. We previously demonstrated that ADAM12 metalloproteinase cleaved ephrin-A1 and ADAM12-cleaved ephrin-A1 enhanced vascular permeability by degrading VE-cadherin and the EphA2 receptor at the plasma membrane. An increase of soluble ephrin-A1 levels in the serum facilitated tumor cell recruitment to the lungs, which resulted in lung metastasis. We also found that ephrin-A1 was overexpressed in 3LL tumors, a highly metastatic tumor, in mice and TNFα, an authentic positive regulator of ephrin-A1, was not elevated in the tumors, whereas S100A8 was. Moreover, S100A8 induced ephrin-A1 expression mediated by the toll-like receptor 4 (TLR4). S100A8 is known to be an endogenous ligand for TLR4 and its expression was shown to be increased in the lungs at the premetastatic phase. Thus, S100A8 and ephrin-A1 contribute to lung metastasis. Therefore, elucidating the regulation mechanism of ephrin-A1 overexpression is of importance and may lead to the development of therapeutic drugs against tumor growth and metastasis.

Serum amyloid A (SAA) 3 is a major component of the acute phase of inflammation. We previously reported that SAA3 served as an endogenous peptide ligand for TLR4 to facilitate lung metastasis. Because these experiments were performed with SAA3 recombinant proteins purified from Escherichia coli or mammalian cells, we could not rule out the possibility of LPS contamination. In this study, we used SAA3 synthetic peptides to eliminate the presence of LPS in SAA3. We found that the SAA3 synthetic peptide (aa 20–86) (20–86) stimulated cell migration and activated p38 in a manner dependent on TLR4, MD-2, and MyD88. SAA3 (20–86) also activated NF-κB and Rho small GTPase. Using surface plasmon resonance analysis, the binding constant KD values between SAA3 (20–86) or SAA3 (43–57) and TLR4/MD-2 protein highly purified by the baculovirus system were 2.2 and 30 μM, respectively. FLAG-tagged SAA3 tightly bound to protein A–tagged MD-2, but not to TLR4 in baculovirus coinfection experiments. Although SAA3 (20–86) caused a low, but appreciable level of endocytosis in TLR4, it induced the upregulation of both IL-6 and TNF-α, but not IFN-β1. An i.v. injection of SAA3 (43–57) induced the lung recruitment of CD11b+Gr-1+ cells at an estimated serum concentration around its KD value toward TLR4/MD-2. Taken together, these results suggest that SAA3 directly binds MD-2 and activates the MyD88-dependent TLR4/MD-2 pathway.

The Eph-ephrin receptor-ligand system is implicated in cell behavior and morphology. EphA1 is the founding member of the Eph receptors, but little is known about its function. Here, we show that activation of EphA1 kinase inhibits cell spreading and migration in a RhoA-ROCK-dependent manner. We also describe a novel interaction between EphA1 and integrin-linked kinase (ILK), a mediator of interactions between integrin and the actin cytoskeleton. The C-terminal sterile α motif (SAM) domain of EphA1 is required and the ankyrin region of ILK is sufficient for the interaction between EphA1 and ILK. The interaction is independent of EphA1 kinase activity but dependent on stimulation of the EphA1 ligand ephrin-A1. Activation of EphA1 kinase resulted in a decrease of ILK activity. Finally, we demonstrated that expression of a kinase-active form of ILK (S343D) rescued the EphA1-mediated spreading defect, and attenuated RhoA activation. These results suggest that EphA1 regulates cell morphology and motility through the ILK-RhoA-ROCK pathway.

Eph receptor tyrosine kinases and their ephrin ligands have been implicated in neuronal development and neovascularization. Overexpression of ephrin-A1 has been implicated in tumor progression and poor prognosis. However, the mechanisms are not clear. Here, we report a role of the Eph/ephrin system in a cell adhesion mechanism. Clustered erythropoietin-producing hepatocellular receptor A1 (EphA1)/ephrin-A1 complexes on the plasma membrane did not undergo endocytosis, and the cell remained adherent to one another. The cell-cell contacts were maintained in an Eph tyrosine kinase activity-independent manner even in the absence of E-cadherin. EphA1 and ephrin-A1 co-localized in pulmonary endothelial cells, and regulated vascular permeability and metastasis in the lungs. We identified ADAM12 (A disintegrin and metalloproteinase 12) as an EphA1-binding partner by yeast two-hybrid screening and found that ADAM12 enhanced ephrin-A1 cleavage in response to transforming growth factor-β1 in primary tumors. Released soluble ephrin-A1 in the serum deteriorated the EphA1/ephrin-A1-mediated cell adhesion in the lungs in an endocrine manner, causing lung hyperpermeability that facilitated tumor cell entry into the lungs. Depletion of soluble ephrin-A1 by its neutralizing antibody significantly inhibited lung metastasis.