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.

Exosome-transmitted S100A4 induces immunosuppression and non-small cell lung cancer development by activating STAT3.

Non-small cell lung cancer (NSCLC) is the primary reason of tumor morbidity and mortality worldwide. We aimed to study the transfer process of S100A4 between cells and whether it affected NSCLC development by affecting STAT3 expression. First, S100A4 expression in NSCLC cells was measured. The exosomes in MRC-5, A549, and H1299 cells were isolated and identified. We constructed si-S100A4 and si-PD-L1 to transfect A549 cells and oe-S100A4 to transfect H1299 cells, and tested the transfection efficiency. Cell function experiments were performed to assess cell proliferation, clone number, apoptosis, cell cycle, migration, and invasion abilities. In addition, ChIP was applied to determine the targeting relationship between S100A4 and STAT3. Next, we explored NSCLC cell-derived exosomes role in NSCLC progress by transmitting S100A4. Finally, we verified the function of exosome-transmitted S100A4 in NSCLC in vivo. High expression of S100A4 was secreted by exosomes. After knocking down S100A4, cell proliferation ability was decreased, clones number was decreased, apoptosis was increased, G1 phase was increased, S phase was repressed, and migration and invasion abilities were also decreased. ChIP validated STAT3 and PD-L1 interaction. After knocking down S100A4, PD-L1 expression was decreased, while ov-STAT3 reversed the effect of S100A4 on PD-L1 expression. Meanwhile, S100A4 inhibited T-cell immune activity by activating STAT3. In addition, knockdown of PD-L1 inhibited cell proliferation, migration, and invasion. NSCLC cell-derived exosomes promoted cancer progression by transmitting S100A4 to activate STAT3 pathway. Finally, in vivo experiments further verified that exosome-transmitted S100A4 promoted NSCLC progression. Exosome-transmitted S100A4 induces immunosuppression and the development of NSCLC by activating STAT3.

[Study of the negative regulation of transforming growth factor beta type II receptor to inhibit the occurrence and development of liver fibrosis with miR-217].

Objective: To observe the effect of miR-217 on angiotensin II (AngII)-induced hepatic stellate cells (HSCs) activation, and carbon tetrachloride (CCl4)-induced overexpression in mice, so as to clarify miR-217 role in liver fibrosis. Methods: HSCs were stimulated with AngⅡ and the changes condition in the expression level of miR-217 were detected. HSCs were divided into control group, AngII-treated group and AngⅡ+miR-217-treated group. The expression levels of alpha-smooth muscle actin, fibroblast-specific protein 1 and collagen Ⅰ (Collagen Ⅰ) in each group were detected. The target gene of mir-217 was screened and verified by Targetscan and Dual luciferase gene reporter assay. Real-time quantitative PCR and Western blot were used to detect the effect of miR-217 on the expression level of transforming growth factor beta type Ⅱ receptor (TGFBR2). A CCl4-induced mouse liver fibrosis model was constructed. Masson staining and Sirius red staining were used to detect the effect of miR-217 overexpression on the progression of liver fibrosis in CCl4 mice. Data of two groups were compared using t-test. Data of multiple groups were statistically analyzed with one-way ANOVA. Results: The expression level of miR-217 was downregulated by AngⅡ-stimulated HSC cells. The expression levels of α-smooth muscle actin, fibroblast-specific protein 1 and Collagen Ⅰ induced by AngⅡ was inhibited by miR-217 mimics transfection. The 3'-UTR of TGFBR2 had specifically bind miR-217. The mRNA and protein expression levels of TGFBR2 was inhibited with miR-217 mimics transfection in HSCs. The overexpression of miR-217 had inhibited the expression levels of Collagen Ⅰ and Ⅲ in CCl4 mice and alleviated the progression of liver fibrosis . Conclusion: miR-217 regulates liver fibrosis by targeting TGFBR2, inhibits AngII-induced HSC activation, and slows down the process of liver fibrosis in CCl4 mice, suggesting that miR-217 may have an inhibitory effect on liver fibrosis.

Neutralization of S100A4 induces stabilization of atherosclerotic plaques: role of smooth muscle cells.

AIMS: During atherosclerosis, smooth muscle cells (SMCs) accumulate in the intima where they switch from a contractile to a synthetic phenotype. From porcine coronary artery, we isolated spindle-shaped (S) SMCs exhibiting features of the contractile phenotype and rhomboid (R) SMCs typical of the synthetic phenotype. S100A4 was identified as a marker of R-SMCs in vitro and intimal SMCs, in pig and man. S100A4 exhibits intra- and extracellular functions. In this study, we investigated the role of extracellular S100A4 in SMC phenotypic transition. METHODS AND RESULTS: S-SMCs were treated with oligomeric recombinant S100A4 (oS100A4), which induced nuclear factor (NF)-κB activation. Treatment of S-SMCs with oS100A4 in combination with platelet-derived growth factor (PDGF)-BB induced a complete SMC transition towards a pro-inflammatory R-phenotype associated with NF-κB activation, through toll-like receptor-4. RNA sequencing of cells treated with oS100A4/PDGF-BB revealed a strong up-regulation of pro-inflammatory genes and enrichment of transcription factor binding sites essential for SMC phenotypic transition. In a mouse model of established atherosclerosis, neutralization of extracellular S100A4 decreased area of atherosclerotic lesions, necrotic core, and CD68 expression and increased α-smooth muscle actin and smooth muscle myosin heavy chain expression. CONCLUSION: We suggest that the neutralization of extracellular S100A4 promotes the stabilization of atherosclerotic plaques. Extracellular S100A4 could be a new target to influence the evolution of atherosclerotic plaques.

S100A4 suppresses cancer stem cell proliferation via interaction with the IKK/NF-κB signaling pathway.

BACKGROUND: Bladder cancer often recurs due to incomplete elimination of the cancer stem cells (CSCs). Therefore, new strategies targeting bladder CSCs are needed and the aim of this study was to investigate the effect of S100A4 on the proliferation capacity of MB49 bladder cancer stem cells (MCSCs). METHODS: MCSCs were established and validated. The expression level of S100A4 in MCSCs and MB49 cells was evaluated using Western blotting and quantitative polymerase chain reaction (QPCR). S100A4 was overexpressed or knocked-down by transfection of pCMV6-XL5-S100A4 plasmid or RNA interference (RNAi) respectively. Proliferation capacity of MCSC was evaluated by cell proliferation assay and in vivo tumorigenicity study. Transcriptional activity of nuclear factor kappa B (NF-κB) was analyzed using luciferase reporter assay, and the level of interleukin (IL)-2 as well as tumor necrosis factor (TNF) was quantified by QPCR. Protein-protein interaction of S100A4 and inhibitor of nuclear factor kappa B NF-κB kinase (IKK) was analyzed by immunoprecipitation. RESULTS: S100A4 was significantly up-regulated in MCSCs, which positively associated with the proliferation capacity, as well as the level of NF-κB, IKK, IL-2 and TNF in MCSCs. Knock-down of S100A4 could reverse such effects. Using immunoprecipitation assay, an interaction between S100A4 and IKK could be observed. CONCLUSIONS: S100A4 is upregulated in MCSCs and possibly enhance the proliferation ability of MCSCs by way of activating the IKK/NF-κB signaling pathway, and S100A4 maybe a hopeful therapeutic target for MCSCs.

Extracellular S100A4 negatively regulates osteoblast function by activating the NF-κB pathway.

Patients with inflammatory bone disease or cancer exhibit an increased risk of fractures and delayed bone healing. The S100A4 protein is a member of the calcium-binding S100 protein family, which is abundantly expressed in inflammatory diseases and cancers. We investigated the effects of extracellular S100A4 on osteoblasts, which are cells responsible for bone formation. Treating primary calvarial osteoblasts with recombinant S100A4 resulted in matrix mineralization reductions. The expression of osteoblast marker genes including osteocalcin and osterix was also suppressed. Interestingly, S100A4 stimulated the nuclear factor-kappaB (NF-κB) signaling pathway in osteoblasts. More importantly, the ex vivo organ culture of mouse calvariae with recombinant S100A4 decreased the expression levels of osteocalcin, supporting the results of our in vitro experiments. This suggests that extracellular S100A4 is important for the regulation of bone formation by activating the NF-κB signaling pathway in osteoblasts. [BMB Reports 2017; 50(2): 97-102].