Downregulation Of S100A4 Research Articles

Anti-S100A4 antibody administration alleviates bronchial epithelial-mesenchymal transition in asthmatic mice.

We elucidated the effect of S100A4 on airway remodeling by regulating airway inflammation and epithelial-mesenchymal transition (EMT) in mouse models of asthma. Asthmatic mouse models were established by sensitization and challenged with ovalbumin (OVA). Anti-S100A4 antibody or control IgG antibody was administered daily before the OVA challenge. After the last challenge, airway inflammation and airway hyperresponsiveness were measured; lung tissues and bronchoalveolar lavage fluid (BALF) were harvested. Lung tissue sections were stained and evaluated for pathological changes. Levels of inflammatory cytokines were measured using ELISA. Levels of S100A4 and EMT markers were determined via western blotting analysis. Human bronchial epithelial cells were stimulated with 100 mg/mL house dust mites (HDMs) to evaluate the effect of S100A4 downregulation on EMT in vitro. S100A4 was increased in lung tissues and BALF from asthmatic mice. The asthmatic mice presented airway hyperresponsiveness, airway inflammation, and airway remodeling. After anti-S100A4 antibody administration, pathophysiological signs, including airway hyperresponsiveness and increased infiltration of inflammatory cells, were attenuated. Additionally, anti-S100A4 administration downregulated vimentin and α-SMA expression and upregulated E-cadherin expression in OVA-challenged mice. S100A4 downregulation also inhibited EMT process in HDM-stimulated 16HBE cells. Anti-S100A4 antibody administration alters airway remodeling by preventing EMT in mouse models of asthma.

Design, synthesis and evaluation of novel Se-alkylated pyrazoles and their cyclized analogs as potential anticancer agents

A simple multicomponent reaction (MCR) was utilized to synthesize a series of 5-alkylated selanyl-1H-pyrazole derivatives 3a-d, 6a-d, and their 4-amino-5-substituted selenolo[2,3-c]pyrazole analogs (4a-d). The methodology is based on the substitution of selenium for the chlorine atom in the chloro pyrazole carbonitrile compound 1via reduction with sodium borohydride in a nitrogen atmosphere, followed by in-situ substitution reactions with various α-halo alkylating agents in the presence of a basic catalyst. The structures of the compounds were confirmed using elemental and spectroscopic techniques (FT-IR, 1H, 13C NMR, and mass spectrometry). The compounds were screened for their anticancer activity which showed promising results and compounds 4c and 6d exhibited the highest cytotoxicity against the HepG2 cell line with pIC50 values of 4.30 and 4.49 respectively, without showing any off-target toxicity. These molecules also, displayed good ADMET features, passed Lipinski's filters, showed good drug-likeness properties, and were predicted to be effective dual EGFR and VEGFR-2 inhibitors, causing downregulation of S100A4 and MMP-9 genes. Together, these results make these molecules as important lead molecules that could be studied further for their anticancer properties.

Clusterin promotes growth and invasion of clear cell renal carcinoma cell by upregulation of S100A4 expression.

Clusterin promotes cell proliferation, motility and invasiveness in human renal cell carcinoma (RCC) cells but the underlying molecular mechanisms of this action are largely unknown. The aim of this study was to investigate the effects of clusterin on cancer cell growth, invasion and S100A4 expression and to determine the effects of clusterin on in vitro cell proliferation and migration and in vivo tumour growth in RCC cells. We have established stable transfectants of highly invasive Caki-1 human RCC cells with expression of clusterin shRNA targeting clusterin (Caki-1/clusterin shRNA). We also established stable transfectants of 786-O human RCC cells with expression of clusterin cDNA plaismid (786-O/clusterin cDNA). Clusterin and S100A4 expression was detected by reverse transcription (RT) PCR and western blot assay; Caki-1/clusterin shRNA and 786-O/clusterin cDNA clones were subjected to in vitro-invasion assays. Cell viability and cell growth was assessed in MTT and clonogenic assay. Specific small interfering RNA was employed to down-regulate S100A4. The expression plasmid for S100A4 (pCMV-S100A4) was used to upregulate S100A4. Caki-1/clusterin shRNA clones were injected subcutaneously in nude mice to determine tumour growth and cancer cell invasiveness in vivo. Xenograft tumour tissues were assessed by immunohistochemistry and frozen tissues were used for the detection of S100A4 and clusterin. Overexpression of clusterin increased cell invasiveness; and targeting clusterin reduced cell invasiveness in vitro. This increase in cell invasiveness was mediated by S100A4. Targeting clusterin decreased cell proliferation and down-regulated cellular S100A4 levels in Caki-1 cells; Overexpression of clusterin increased cell proliferation and up-regulated cellular S100A4 levels in 786-O cells; Stable Caki-1/clusterin shRNA transfectants produced smaller xenograft tumours containing reduced S100A4 protein levels in vivo. Stable 786-O/clusterin cDNA transfectants produced larger xenograft tumours containing increased S100A4 protein levels in vivo. Our results indicate that clusterin promotes growth and invasion in RCC cells in vitro and in vivo through upregulation of S100A4; And targeting clusterin confers growth inhibitory and anti-invasive properties in RCC cells in vitro and in vivo through a down-regulation of S100A4. These findings provide the rationale for future oncostatic strategies aimed at suppressing clusterin-mediated signal transduction pathways as a novel therapeutic approach in human RCC.

Downregulation of S100A4 Alleviates Cardiac Fibrosis via Wnt/β -Catenin Pathway in Mice

Background/Aims: Cardiac fibrosis is a pathological change leading to cardiac remodeling during the progression of myocardial ischemic diseases, and its therapeutic strategy remains to be explored. S100A4, a calcium-binding protein, participates in fibrotic diseases with an unclear mechanism. This study aimed to investigate the role of S100A4 in cardiac fibrosis. Methods: Cardiac fibroblasts from neonatal C57BL/6 mouse hearts were isolated and cultured. Myocardial infarction was induced by ligating the left anterior descending coronary artery (LAD). The ligation was not performed in the sham group. A volume of 5×10⁵pfu/g adenovirus or 5 µM/g ICG-001 was intramyocardially injected into five parts bordering the infarction zone or normal region. We used Western blotting, quantitative RT-PCR, immunofluorescence, immunohistochemistry and Masson’s trichrome staining to explore the function of S100A4. Results: We found significant increases of S100A4 level and cardiac fibrosis markers, and β-catenin signaling activation in vitro and in vivo. In addition, knockdown of S100A4 significantly reduced cardiac fibrosis and β-catenin levels. Moreover, the expression of S100A4 decreased after ICG-001 inhibited β-catenin signal pathway. Conclusion: Downregulation of S100A4 alleviates cardiac fibrosis via Wnt/β -catenin pathway in mice. S100A4 may be a therapeutic target of cardiac fibrosis.

Reduction in Migratory Phenotype in a Metastasized Breast Cancer Cell Line via Downregulation of S100A4 and GRM3

To investigate phenotypic and genotypic alterations before and after bone metastasis, we conducted genome-wide mRNA profiling and DNA exon sequencing of two cell lines (TMD and BMD) derived from a mouse xenograft model. TMD cells were harvested from the mammary fat pad after transfecting MDA-MB-231 breast cancer cells, while BMD cells were isolated from the metastasized bone. Compared to BMD cells, TMD cells exhibited higher cellular motility. In contrast, BMD cells formed a spheroid with a smoother and more circular surface when co-cultured with osteoblasts. In characterizing mRNA expression using principal component analysis, S100 calcium-binding protein A4 (S100A4) was aligned to a principal axis associated with metastasis. Partial silencing of S100A4 suppressed migratory capabilities of TMD cells, while Paclitaxel decreased the S100A4 level and reduced TMD’s cellular motility. DNA mutation analysis revealed that the glutamate metabotropic receptor 3 (GRM3) gene gained a premature stop codon in BMD cells, and silencing GRM3 in TMD cells altered their spheroid shape closer to that of BMD cells. Collectively, this study demonstrates that metastasized cells are less migratory due in part to the post-metastatic downregulation of S100A4 and GRM3. Targeting S100A4 and GRM3 may help prevent bone metastasis.

Molecular inhibition mechanisms of cell migration and invasion by coix polysaccharides in A549 NSCLC cells via targeting S100A4.

S100 calcium binding protein A4 (S100A4) promotes extracellular signal transduction, intercellular adhesion, motility and mobility. Different extracts from Coixlachryma-jobi have been used for the treatment of various types of cancer in Asia. In our previous study, the polysaccharide fraction extact, CP1, induced cell apoptosis of non‑small cell lung cancer cells. In the current study, CP1 inhibited migration and invasion of A549 cells in a scratch wound healing assay and matrigel invasion assay, respectively. Furthermore, reverse transcription‑polymerase chain reaction and western blotting demonstrated that CP1 downregulated the gene and protein expression levels of S100A4. In silico docking analysis demonstrated that polysaccharides may not interfere with dimerization, whereas, the affinity of polysaccharides for an S100A4‑NMIIA pocket was margnially greater than at the dimerization sites. Thus, CP1 inhibited A549 cell migration and invasion potentially via downregulation of S100A4, and may also interact with the binding site of S100A4‑NMIIA, which indicated that CP1 has potential as an alternative cancer chemotherapeutic by targeting S100A4.

Small interfering RNA targeting S100A4 sensitizes non-small-cell lung cancer cells (A549) to radiation treatment.

ObjectiveThis study aimed to investigate the impact of S100A4-small interfering RNA (S100A4-siRNA) on apoptosis and enhanced radiosensitivity in non-small-cell lung cancer (A549) cells. We also explored the mechanisms of radiosensitization and identified a new target to enhance radiosensitivity and gene therapy for non-small-cell lung cancer.MethodsRNA interference is a powerful tool for gene silencing. In this study, we constructed an effective siRNA to knock down S100A4. A549 cells were randomly divided into three groups: blank, negative control, and S100A4-siRNA. To investigate the effect of S100A4-siRNA, the expression of S100A4, E-cadherin, and p53 proteins and their messenger RNA (mRNA) was detected by Western blot and quantitative real-time polymerase chain reaction. Transwell chambers were used to assess cell invasion. Cell cycle and apoptosis were analyzed by flow cytometry. Radiosensitivity was determined by colony formation ability.ResultsOur results demonstrate that S100A4-siRNA effectively silenced the S100A4 gene. When siRNA against S100A4 was used, S100A4 protein expression was downregulated, whereas the expressions of E-cadherin and p53 were upregulated. In addition, a clear reduction in S100A4 mRNA levels was noted compared with the blank and negative control groups, whereas E-cadherin and p53 mRNA levels increased. Transfection with S100A4-siRNA significantly reduced the invasiveness of A549 cells. S100A4 silencing induced immediate G2/M arrest in cell cycle studies and increased apoptosis rates in A549 cells. In clonogenic assays, we used a multitarget, single-hit model to detect radiosensitivity after S100A4 knockdown. All parameters (D0, Dq, α, β) indicated that the downregulation of S100A4 enhanced radiosensitivity in A549 cells. Furthermore, S100A4-siRNA upregulated p53 expression, suggesting that S100A4 may promote A549 cell proliferation, invasion, and metastasis by regulating the expression of other proteins. Therefore, siRNA-directed S100A4 knockdown may represent a viable clinical therapy for lung cancer.ConclusionS100A4 downregulation potentially enhances the sensitivity of human A549 cells to radiotherapy.

STAT1 modification improves therapeutic effects of interferons on lung cancer cells.

BackgroundInterferons (IFNs) have potent anti-proliferative, pro-apoptotic, and immunomodulatory activities against cancer. However, the clinical utility of IFNs is limited by toxicity and pharmacokinetics making it difficult to achieve sustained therapeutic levels especially in solid tumors.MethodsSignal Transducer and Activator of Transcription 1 (STAT1) or a modified STAT1 (designated STAT1-CC) that is hyper-responsive to IFN were overexpressed in lung cancer SPC-A-1 and H1299 cells using lentiviral vectors. Transduction efficiency was monitored using enhanced green fluorescent protein (EGFP) expression. After transduction, cells were treated with interferon-gamma (IFN-γ) or interferon-beta (IFN-β) and monitored for cell proliferation, migration, and invasiveness using Cell Counting Kit-8 and transwell chamber assays and for apoptosis using Annexin V detection by flow cytometry. In addition, levels of STAT1, STAT1 Tyr-701 phosphorylation (pSTAT1), fibronectin, and β-catenin were determined using western blotting. In the case of IFN-γ stimulation, levels of S100A4, proliferating cell nuclear antigen (PCNA), and c-fos expression were also determined.ResultsWe found that expression of STAT1 or STAT1-CC enhanced the effect of IFN-γ and, IFN-β on inhibition of human lung cancer cell proliferation, migration and invasiveness. Moreover, STAT1 and STAT1-CC expression caused increases in pSTAT1 and decreases in fibronectin and β-catenin levels. STAT1-CC showed increased effects compared to STAT1 on IFN-γ induced pSTAT1 and down-regulation of S100A4, PCNA, and c-fos levels.ConclusionThe results show that STAT1-CC exhibited more strength in improving the antitumor response of IFNs in lung cancer cells. Results from this study suggest that combined treatment of IFNs and STAT1-CC might be a feasible approach for the clinical management of lung cancer in the future.

Knockdown of S100A4 impairs arecoline-induced invasiveness of oral squamous cell carcinomas

Metastasis is the most common cause of oral squamous cell carcinoma (OSCC)-related death. The physiological function of S100A4 in the pathogenesis of areca quid chewing-associated OSCC has not been uncovered. OSCC tissues from areca quid chewers were analyzed by immunohistochemistry for S100A4 expression. The functions of S100A4 in invasiveness of arecoline-treated oral epithelial (OE) cells were determined by loss function approaches. Expression of S100A4 was positively correlated with clinical grading and lymph node metastasis of OSCC. Upregulated S100A4 is correlated with poor survival outcome of OSCC patients. Arecoline led to dose-dependent elevation of S100A4 expression in oral epithelial (OE) cells. Down-regulation of S100A4 significantly reversed arecoline-induced oncogenecity in OE cells. The additions of pharmacological agents LY294002, SP600125, and CAY10585 were found to inhibit arecoline-induced S100A4 expression in OE cells. Arecoline-induced S100A4 expression was down-regulated by LY294002, SP600125, or CAY10585 treatment. Targeting S100A4 might offer a new strategy for the treatment of OSCC patients with metastasis.

S100A4 promotes pancreatic cancer progression through a dual signaling pathway mediated by Src and focal adhesion kinase.

S100A4 expression is associated with poor clinical outcomes of patients with pancreatic cancer. The effects of loss or gain of S100A4 were examined in pancreatic cancer cell lines. S100A4 downregulation remarkably reduces cell migration and invasion, inhibits proliferation, and induces apoptosis in pancreatic tumor cells. S100A4 downregulation results in significant cell growth inhibition and apoptosis in response to TGF-β1, supporting a non-canonical role of S100A4 in pancreatic cancer. The role of S100A4 in tumor progression was studied by using an orthotopic human pancreatic cancer xenograft mouse model. Tumor mass is remarkably decreased in animals injected with S100A4-deficient pancreatic tumor cells. P27Kip1 expression and cleaved caspase-3 are increased, while cyclin E expression is decreased, in S100A4-deficient pancreatic tumors in vivo. S100A4-deficient tumors have lower expression of vascular endothelial growth factor, suggesting reduced angiogenesis. Biochemical assays revealed that S100A4 activates Src and focal adhesion kinase (FAK) signaling events, and inhibition of both kinases is required to maximally block the tumorigenic potential of pancreatic cancer cells. These findings support that S100A4 plays an important role in pancreatic cancer progression in vivo and S100A4 promotes tumorigenic phenotypes of pancreatic cancer cells through the Src-FAK mediated dual signaling pathway.

Neutrophils Promote the Malignant Glioma Phenotype through S100A4

Antiangiogenic therapy is effective in blocking vascular permeability, inhibiting vascular proliferation, and slowing tumor growth, but studies in multiple cancer types have shown that tumors eventually acquire resistance to blockade of blood vessel growth. Currently, the mechanisms by which this resistance occurs are not well understood. In this study, we evaluated the effects of neutrophils on glioma biology both in vitro and in vivo and determined target genes by which neutrophils promote the malignant glioma phenotype during anti-VEGF therapy. We found that an increase in neutrophil infiltration into tumors is significantly correlated with glioma grade and in glioblastoma with acquired resistance to anti-VEGF therapy. Our data demonstrate that neutrophils and their condition media increased the proliferation rate of glioblastoma-initiating cells (GIC). In addition, neutrophils significantly increased GICs Transwell migration compared with controls. Consistent with this behavior, coculture with neutrophils promoted GICs to adopt morphologic and gene expression changes consistent with a mesenchymal signature. Neutrophil-promoting tumor progression could be blocked by S100A4 downregulation in vitro and in vivo. Furthermore, S100A4 depletion increased the effectiveness of anti-VEGF therapy in glioma. Collectively, these data suggest that increased recruitment of neutrophils during anti-VEGF therapy promotes glioma progression and may promote treatment resistance. Tumor progression with mesenchymal characteristics is partly mediated by S100A4, the expression of which is increased by neutrophil infiltration. Targeting granulocytes and S100A4 may be effective approaches to inhibit the glioma malignant phenotype and diminish antiangiogenic therapy resistance.

Spatiotemporal Expression of Matrix Metalloproteinases (MMPs) is Regulated by the Ca2+-Signal Transducer S100A4 in the Pathogenesis of Thoracic Aortic Aneurysm

AimsThis study investigated whether S100A4 plays a potential role in the formation of thoracic aortic aneurysm (TAA).Methods and ResultsThe thoracic aortas of male Sprague-Dawley rats were exposed to 0.5 M CaCl2 or normal saline (NaCl). Animals were euthanized at specified time-points (2, 4, and 10 weeks post-TAA induction). The treated aortic segments were harvested, and mRNA levels, protein expressions and immunohistochemistry of MMP-2, MMP-9 and S100A4 were analyzed. The A7r5 cell lines were used for an in vitro study. Experiments were also performed using human TAA samples for comparison. Localized aneurysmal dilation was observed in the CaCl2-treated segments. The transcription levels of S100A4 and MMPs were elevated in CaCl2-treated segments versus controls, and a significant correlation between S100A4 and expression of MMPs was observed across all time-points. Immunohistochemical studies revealed similar expression pattern of S100A4 and MMP proteins, as well as co-localization of S100A4 with the cell lineage markers (αSMA and CD68) and inflammatory markers (MMPs and NF-κB P65 subunit). The proliferative ability of A7r5 cells after transfection with S100A4 siRNA was suppressed, and down-regulation of S100A4 inhibited MMP-2 and MMP-9 expression in vitro. Increased expression of S100A4 was observed in all layers of the aorta wall in human TAA specimens. Serum concentrations of S100A4 determined by ELISA were found to be significantly increased in TAA patients.ConclusionsThis study established the important roles of S100A4 and MMPs in the development of TAA.

Elevation of S100A4 Expression in Buccal Mucosal Fibroblasts by Arecoline: Involvement in the Pathogenesis of Oral Submucous Fibrosis

BackgroundS100A4, a member of the calcium-binding proteins, is dramatically elevated in a variety of fibrotic diseases. Areca quid chewing is the most important etiological factor in the pathogenesis of oral submucous fibrosis (OSF). OSF has been considered as a pre-cancerous condition of oral mucosa. The aim of this study was to determine the critical role of S100A4 expression in the pathogenesis of OSF both in vitro and in vivo.Methodology/Principal FindingThirty OSF tissues from areca quid chewers and ten normal buccal mucosa samples without areca quid chewing were analyzed by using immunohistochemistry for S100A4 expression in vivo. Collagen gel contraction capability and expression of tissue inhibitor of metalloproteinases 1 (TIMP1)/MMP9 in arecoline-stimulated BMFs with S100A4 knockdown was presented in vitro. Initially, S100A4 expression was higher in areca quid chewing-associated OSF specimens than normal buccal mucosa specimens (p = 0.001). Arecoline, a major areca nut alkaloid, led to dose- and time-dependent elevation of S100A4 expression in normal buccal mucosa fibroblasts BMFs (p<0.05). The additions of pharmacological agents rapamycin (mTOR inhibitor), PD98059 (ERK inhibitor), and Bay117082 (NF-κB inhibitor) were found to inhibit arecoline-induced S100A4 expression (p<0.05) in BMFs. Down-regulation of S100A4 by lentiviral infection significantly reversed arecoline-induced collagen gel contraction and TIMP1/MMP9 expression.Conclusion/SignificanceThese results suggest that S100A4 expression is significantly up-regulated in OSF specimens. Arecoline-induced S100A4 expression was down-regulated by rapamycin, PD98059, and Bay117082. Targeting S100A4 might be a potential therapeutic target for OSF through TIMP1/MMP9 down-regulation.

Abstract 97: Spatiotemporal Expression Of Matrix Metalloproteinases (mmps) Is Regulated By Ca2+-signal Transducer S100a4 In The Thoracic Aortic Aneurysm Pathogenesis

Purpose: Ca 2+ signaling pathways play critical roles in a wide range of physiological functions including control and regulation of smooth muscle cell performance and vascular tone. Previous studies have indicated that Ca 2+ signaling transducer S100A4 could increase the secretion of MMPs from endothelial cells and fibroblasts and is greatly expressed in smooth muscle cells in atherosclerotic lesions. This study investigated whether S100A4 has a potential role in the formation of TAA. Methods: Thoracic aorta of male Sprague-Dawley rats was exposed to 0.5 M CaCl 2 or normal saline (NaCl). Animals were euthanized at specified time points (2, 4, 10 weeks post-TAA induction). The treated aortic segments were harvested, and mRNA levels, protein expressions and immunohistochemistry regarding MMP-2, MMP-9 and S100A4 were analyzed. The A7r5 cell lines were used as in vitro study. Experiments were also performed using human TAA samples for comparison. Results: Localized aneurysmal dilation was observed in the CaCl 2 -treated segments, with disruption of elastic lamellae and decrease of intima-media thickness (minimum at 2 weeks). The transcription levels of S100A4 and MMPs were elevated in CaCl 2 -treated segments versus controls (most prominent at 2 weeks), and a significant correlation between S100A4 and MMPs expression was observed across the time points (MMP-2, r=0.652, p&lt;0.05; MMP-9, r=0.762, p&lt;0.05). Immunohistochemistry studies have revealed similar expression pattern of S100A4 and MMPs proteins, as well as co-localization of S100A4 with the cell lineage markers (αSMA and CD68) and inflammatory markers (MMPs and NF-κB P65 subunit). Proliferation ability of A7r5 cells after transfection with S100A4 siRNA was suppressed and down regulation of S100A4 inhibited MMP-2 expression in vitro . Increased expression of S100A4 was observed in the entire layers of aorta wall in human TAA specimens. And serum concentrations of S100A4 determined by ELISA were found significantly increased in TAA patients. Conclusion: This study established an important role of S100A4 with MMPs in the development of TAA. S100A4 may serve as a biomarker for TAA diagnosis and a potential target for therapy.

Downregulation of S100A4 expression by RNA interference suppresses cell growth and invasion in human colorectal cancer cells

S100A4 protein, a member of the S100 superfamily of calcium-binding proteins, is frequently observed in various types of human cancers, including colorectal cancer (CRC). Our previous investigations have demonstrated that the overexpression of S100A4 is associated with lymph node metastasis and poor prognosis in CRC; however, its biological roles in CRC remain unclear. In the present study, we compared the expression of S100A4 at the mRNA and protein levels in six CRC cell lines, and found that the expression levels roughly coincided with their invasiveness. Using RNA interference, we suppressed S100A4 expression in SW620 CRC cells with highly invasive potential and S100A4 high expression. The specific knockdown of S100A4 strongly suppressed cell growth, migration and invasion activities. Furthermore, employing metastasis-related gene mRNA microarrays, we found four genes to be significantly dysregulated (more than 2-fold) after downregulation of S100A4, including three downregulated genes (MMP9, MMP10 and CDH11) and one upregulated gene (TIMP4). Our present results indicate that S100A4 may positively regulate tumor cell proliferation, invasion and metastasis associated with multiple molecules. Thus, the inhibition of S100A4 might be a potentially novel approach to treatment for CRC.

The metastasis-promoting protein S100A4 regulates mammary branching morphogenesis

High levels of the S100 calcium binding protein S100A4 also called fibroblast specific protein 1 (FSP1) have been established as an inducer of metastasis and indicator of poor prognosis in breast cancer. The mechanism by which S100A4 leads to increased cancer aggressiveness has yet to be established; moreover, the function of this protein in normal mammary gland biology has not been investigated. To address the role of S100A4 in normal mammary gland, its spatial and temporal expression patterns and possible function in branching morphogenesis were investigated. We show that the protein is expressed mainly in cells of the stromal compartment of adult humans, and during active ductal development, in pregnancy and in involution of mouse mammary gland. In 3D culture models, topical addition of S100A4 induced a significant increase in the TGFα mediated branching phenotype and a concomitant increase in expression of a previously identified branching morphogen, metalloproteinase-3 (MMP-3). These events were found to be dependent on MEK activation. Downregulation of S100A4 using shRNA significantly reduced TGFα induced branching and altered E-cadherin localization. These findings provide evidence that S100A4 is developmentally regulated and that it plays a functional role in mammary gland development, in concert with TGFα by activating MMP-3, and increasing invasion into the fat pad during branching. We suggest that S100A4-mediated effects during branching morphogenesis provide a plausible mechanism for how it may function in breast cancer progression.

Abstract 4263: Epithelial-mesenchymal transition mediator, S100A4, in maintenance of head and neck cancer initiating cells

Abstract Background: Cancer initiating cells (CICs) represent a rare subpopulation of cells, which are responsible for tumor growth. Previously, we have identified and characterized the CICs from head and neck squamous cell carcinomas (HNSCCs), termed head and neck cancer initiating cells (HN-CICs). HN-CICs possess both the hallmarks of stem cells and malignant tumor cells. Herein, we determined the critical role of S100A4, a common mediator of epithelial-mesenchymal transition (EMT), in maintenance of stemness properties and cancerous phenotypes in HN-CICs. Methods: Affymetrix microarray analysis was performed to identify S100A4 as a putative candidate on mediating the stemness properties of HN-CICs. Consequently, we elucidated the stemness properties and tumorigenicity of HN-CICs with S100A4 down-regulation or overexpression, respectively. Finally, immunohistochemical staining of S100A4 on HNSCC tumor tissues was examined. Results: S100A4 transcripts and protein level were found significantly up regulated in the enriched HN-CICs. Down-regulation of S100A4 significantly reduced the self-renewal ability and stemness properties in HNSCCs. In addition, knockdown of S100A4 lessened tumorigenicity of HNSCCs both in vitro and in vivo. Conversely, over-expression of S100A4 enhanced the CSC properties in HNSCCs. Furthermore, expression of S100A4 was positively correlated with clinical grading, and survivals and recurrence, respectively (p&amp;lt;0.001, 0.05 and 0.01) according to the immunohistochemical staining. Finally, depletion of endogenous S100A4 resulted in down-regulation of Notch2 and PI3K/pAKT and up-regulation of PTEN in HNSCCs. Conclusion: Our study reveals the crucial role of S100A4 signaling pathways in the maintenance of stemness properties and tumorigenicity of HN-CICs, and S100A4 might be a potential therapeutic target for HNSCC by eliminating HN-CICs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4263.

Methylation‐associated silencing of S100A4 expression in human epidermal cancers

S100A4 appears important for cancer metastasis and its overexpression is common in a variety of human malignancies, but its status in epidermal cancers remains lesser known. Likewise, E-cadherin downregulation and Wingless (Wnt) activation are frequent cancer-associated alterations, whereas their potential correlations with S100A4 expression in skin lesions have not been characterized. These issues were addressed in the present study using tissue microarray-based immunohistochemical staining, reverse transcriptase polymerase chain reaction and western blotting. Meanwhile, the underlying epigenetic mechanism leading to the altered S100A4 expression in epidermal tumors was elucidated. Immunohistochemistry revealed that S100A4 expression frequencies were 100% (8/8) in normal epidermis, 80.6% (25/31) in tumor-surrounding non-cancerous epidermis, 66.7% (10/15) in premalignant diseases, 8.3% (1/11) in Bowen's disease and 7.7-26.3% in different cancer tissues. The incidence of S100A4 detection in the normal and non-cancerous epidermis was significantly different from that of epidermal cancers (P = 0.000). Accordingly, human immortalized keratinocyte line HaCat but not skin squamous cell carcinoma (SCC) line colo16 was positive in S100A4 expression. S100A4 downregulation, E-cadherin reduction and Wnt activation coexisted in most of epidermal cancers but unnecessarily overlapped. Methylation DNA sequencing revealed methylation of four critical (cytosine and guanine separated by a phosphate or -C-phosphate-G-) CpG sites within S100A4 intron first in S100A4-negative colo16 cells and skin SCCs, and demethylator/5-aza-2'-deoxycytidine treatment efficiently recovered S100A4 expression in colo16 cells. Our findings demonstrate that S100A4 downregulation, as the consequence of DNA methylation, is closely correlated with skin tumor formation. Wnt activation and E-cadherin reduction and S100A4 down-regulation are paralleled molecular events in skin tumors, which may serve as the biomarkers for predicting epidermal cancer risk.

Relaxin reduces xenograft tumour growth of human MDA-MB-231 breast cancer cells

IntroductionRelaxin levels are increased in cases of human breast cancer and has been shown to promote cancer cell migration in carcinoma cells of the breast, prostate gland and thyroid gland. In oestrogen receptor alpha-negative MDA-MB-231 human breast cancer cells, relaxin was shown to down-regulate the metastasis-promoting protein S100A4 (metastasin), a highly significant prognostic factor for poor survival in breast cancer patients. The cellular mechanisms of relaxin exposure in breast cancer cells are not fully understood. The aim of this study was to investigate short-term and long-term effects of relaxin on cancer cell motility and S100A4 expression and to determine the long-term effects of relaxin on in vivo tumour growth in an oestrogen-independent context.MethodWe have established stable transfectants of highly invasive oestrogen-receptor alpha-negative MDA-MB-231 human breast cancer cells with constitutive expression of bioactive H2-relaxin (MDA/RLN2). RLN2 secretion was determined by ELISA. Relaxin receptor RXFP1 (Relaxin-family-peptide) was detected by reverse transcription (RT) PCR and its activation was assessed by induction of cyclic adenosine monophosphate (cAMP). Stable MDA/RLN2 clones and RLN2 treated MDA-MB-231 cells were subjected to motility and in vitro-invasion assays. Proliferation was assessed in bromodeoxyuridine (BrdU) and MTT assays. S100A4 expression was determined by RT-PCR and Western blot. Specific small interfering RNA was employed to down-regulate relaxin receptor and S100A4. MDA/EGFP vector control and two MDA/RLN2 clones were injected subcutaneously in nude mice to determine tumour growth and cancer cell invasiveness in vivo. Xenograft tumour tissues were assessed by histology and immunohistochemistry and frozen tissues were used for the detection of S100A4 and RLN2.ResultsShort-term exposure to relaxin for 24 hours increased cell motility in a relaxin receptor-dependent manner. This increase in cell motility was mediated by S100A4. Long-term exposure to relaxin secreted from stable transfectants reduced cell motility and in vitro invasiveness. Relaxin decreased cell proliferation and down-regulated cellular S100A4 levels in MDA-MB-231 and T47D breast cancer cells. Stable MDA/RLN2 transfectants produced smaller xenograft tumours containing reduced S100A4 protein levels in vivo.ConclusionOur results indicate that long-term exposure to relaxin confers growth inhibitory and anti-invasive properties in oestrogen-independent tumours in vivo, which may in part be mediated through a down-regulation of S100A4.

Intracellular calcium-binding protein S100A4 influences injury-induced migration of white matter astrocytes

Astrocytes play a crucial role in central nervous system (CNS) pathophysiology. White and gray matter astrocytes are regionally specialized, and likely to respond differently to CNS injury and in CNS disease. We previously showed that the calcium-binding protein S100A4 is exclusively expressed in white matter astrocytes and markedly up-regulated after injury. Furthermore, down-regulation of S100A4 in vitro significantly increases the migration capacity of white matter astrocytes, a property, which might influence their function in CNS tissue repair. Here, we performed a localized injury (scratch) in confluent cultures of white matter astrocytes, which strongly express S100A4, and in cultures of white matter astrocytes, in which S100A4 was down-regulated by transfection with short interference (si) S100A4 RNA. We found that S100A4-silenced astrocytes rapidly migrated into the injury gap, whereas S100A4-expressing astrocytes extended hypertrophied processes toward the gap, but without closing it. To explore the involvement of S100A4 in migration of astrocytes in vivo, we induced focal demyelination and transient glial cell elimination in the spinal cord white matter by ethidium bromide injection in S100A4 (-/-) and (+/+) mice. The results show that astrocyte migration into the demyelinated area is promoted in S100A4 (-/-) compared to (+/+) mice, in which a pronounced glial scar was formed. These data indicate that S100A4 reduces the migratory capacity of reactive white matter astrocytes in the injured CNS and is involved in glial scar formation after injury.