6275

S100A4

S100 calcium binding protein A4;S100A4;S100 calcium binding protein A4

S100 calcium-binding protein A4 (calcium protein, calvasculin, metastasin, murine placental homolog)|fibroblast-specific protein-1|leukemia multidrug resistance associated protein|malignant transformation suppression 1|placental calcium-binding protein|pr

1q21

protein-coding

BACKGROUND: S100A4 gene product has been implicated in tumor invasion and metastasis. The overall survival rate of children with neuroblastoma remains poor because of disease dissemination at the time of diagnosis. The purpose of this study was to investigate the effect and mechanism of S100A4 on invasion and metastasis of neuroblastoma. METHODS: A 20-mer phosphorothioate antisense oligodeoxynucleotide (asODN) targeted against the S100A4 mRNA was transfected into the human neuroblastoma cell line LA-N-6 by Lipofectamine 2000. The expressions of S100A4 and MMP-2 mRNAs were quantified by the reverse transcription polymerase chain reaction. The capability of migration and invasion of LA-N-6 cells were evaluated by the transwell chamber assay. RESULTS: The S100A4 mRNA and the MMP-2 mRNA levels in asODN-treated cells were decreased by 35.6% and 25.5%, respectively, compared with those in nontreated cells. The numbers of migrating and invading LA-N-6 cells were both significantly lower in the asODN-treated groups than those in the nontreated groups ( 9.33 +/- 4.73 vs 20.67 +/- 2.89 and 2.33 +/- 1.15 vs 9.00 +/- 2.65, respectively; both P = .03 ). CONCLUSIONS: The S100A4 asODN significantly reduced the S100A4 mRNA levels and the motility and invasive ability of neuroblastoma cells, with concomitant decrease of the MMP-2 mRNA levels. Thus, S100A4 may exert its effect on invasion and metastasis of neuroblastoma cells by stimulating the motility of tumor cells as well as influencing the expression of MMP-2.

S100A4 is a member of the S100 family of calcium-binding proteins that is directly involved in tumor metastasis. In the present study, we examined the potential role of S100A4 in metastasis in breast cancer and its relation with matrix metalloproteinase-13 (MMP-13). Analysis of 100 breast cancer specimens including 50 with and 50 without lymph node metastasis showed a significant upregulation of S100A4 and MMP-13 expression in metastatic breast cancer tissues. Positive immunoreactivity for S100A4 was associated with MMP-13 expression. Overexpression of S100A4 in the MDA-MB-231 breast cancer cell line upregulated MMP13 expression leading to increased cell migration and angiogenesis. SiRNA-mediated silencing of S100A4 downregulated MMP13 expression and suppressed cell migration and angiogenesis. Moreover, neutralization of MMP-13 activity with a specific antibody blocked cell migration and angiogenesis in MDA-MB-231/S100A4 cells. In vivo siRNA silencing of S100A4 significantly inhibited lung metastasis in transgenic mice. The present results suggest that the S100A4 gene may control the invasive potential of human breast cancer cells by modulating MMP-13 levels, thus regulating metastasis and angiogenesis in breast tumors. S100A4 could therefore be of value as a biomarker of breast cancer progression and a novel therapeutic target for human breast cancer treatment.

OBJECTIVES: To investigate levels of S100A4 protein in plasma and S100A4 mRNA in tumours from patients with clear cell renal cell carcinoma (CCRCC), and correlate these with metastasis, survival and levels of vascular endothelial growth factor (VEGF). METHODS: Plasma S100A4 and VEGF protein concentrations were measured using enzyme-linked immuno sorbent assays in 39 healthy subjects and 68 consecutive patients with untreated CCRCC. Levels of S100A4 and VEGF mRNA in tumour and matched control (healthy) tissue samples were measured using realtime quantitative reverse transcription- polymerase chain reaction. Findings were analysed with respect to clinico pathological characteristics. RESULTS: Plasma VEGF concentrations were higher in patients with CCRCC than in healthy subjects. S100A4 and VEGF mRNA levels were up-regulated in CCRCC tumour tissue compared with control tissue samples. Logistic regression analysis revealed that up-regulated tumour S100A4 and VEGF mRNA levels were independent risk factors for the presence of invasion and/or metastasis. CONCLUSIONS: S100A4 and VEGF are associated with tumour invasion and metastasis, and may be useful prognostic markers in patients with CCRCC. S100A4 and VEGF may represent potential targets for therapeutic intervention.

OBJECTIVE: S100A6 and S100A4, two of S100 protein family, have been suggested to be associated with cancer tumorigenesis and metastasis. The aim of this study was to evaluate the expression levels of S100A6 and S100A4 in matched samples of primary human colorectal adenocarcinomas (T), adjacent normal colorectal mucosa (N) and liver metastases (M). This gave us the advantage of directly comparing levels of S100A6 and S100A4 expression within the same genetic background. METHODS: In matched samples of N, T and M from 10 colorectal adenocarcinoma patients, expressions of S100A6 and S100A4 were studied by Western blot and immunohistochemical analyses using specific antibodies against each protein. RESULTS: The expression levels of S100A6 were significantly higher in T than in N (p < 0.05), while those of S100A4 showed no difference between T and N. There were no significant differences in the expression levels of S100A6 or S100A4 between M and T. Similar results were obtained by immunohistochemical analysis. Moreover, S100A6 was stained more intensely in invading fronts than in central portions of both T and M. CONCLUSIONS: The observed differential expression of S100A6 and S100A4 suggests that S100A6, rather than S100A4, is associated with human colorectal adenocarcinoma tumorigenesis and invasion/metastasis.

To investigate the expression levels of S100A4 in human colorectal carcinoma (CC) and its relationship with clinicopathological parameters and metastatic potential, 73 pathological specimens from patients with CC were examined for S100A4 expression by RT-PCR and immunohistochemistry. An increase of S100A4 mRNA was observed in 19/23 (82.6%) CC specimens, and S100A4 was up-regulated in 40/73 (54.7%) CC cases compared with non-neoplastic mucosal tissues. Upregulation of S100A4 was significantly related to invasion, nodal status, distant metastasis and p53 expression. Next, we investigated whether S100A4 could affect p53 transactivation and stability. Interestingly, it was revealed that treatment with exogenous S100A4 protein reduced transcriptional activity of p53 and abrogated the modification of calcium binding affinity of S100A4 protein. These findings suggested that S100A4 might be involved in the progression and metastasis of human CC, presumably via modulation of the wild-type p53 protein.

To investigate whether S100A14 and S100A4 expression correlates with metastatic potential and prognosis in colorectal cancer (CRC), we firstly used RT-PCR analysis to detect mRNA expression of S100A14 and S100A4 in 40 pairs of fresh tumor samples matched with adjacent normal tissues. We then evaluated the clinical significance of our findings with immunohistochemistry on 115 samples of formalin-fixed and paraffin-embedded tumors on tissue microarrays. Typically, we identified decreased S100A14 mRNA levels (52.5%, 21/40), and increased S100A4 mRNA levels (70.0%, 28/40) in primary CRC samples. In addition, down-regulated or absent S100A14 expression was detected in 56.5% of samples (65/115) and was correlated with poor differentiation (P=0.010). In contrast, overexpressed S100A4 was detected in 57.4% of samples (66/115) and was associated with lymph node metastasis (P=0.001). Simultaneous S100A14 low-expression and S100A4 high-expression was correlated with high CRC metastatic potential (P<0.001). Taken together, the signature derived from the combined expression status of S100A14 and S100A4 could be a valuable prognostic indicator in CRC.

Colon cancer is still a burden mainly due to metastasis formation. The latter is often associated with a constitutive activation of the Wnt/beta-catenin signaling pathway and high expression of the metastasis-inducing gene S100A4. We previously identified S100A4 as a transcriptional target of beta-catenin. Intervention strategies targeting Wnt/beta-catenin signaling might therefore represent promising approaches to inhibit tumor growth and metastasis formation when induced by S100A4. Many inhibitors, various strategies, as well as different routes of application targeting key molecules of the Wnt signaling pathway have been reported within the last decade. Consequently, downregulation of beta-catenin target genes lead to altered tumorigenic and metastastic abilities of cancer cells. This review focuses on the potential of Wnt/beta-catenin signaling intervention to restrict colon cancer metastasis formation by interdicting S100A4 expression.

AIM: To develop lymph node metastasis (LNM)-associated biomarkers for colorectal cancer (CRC) using quantitative proteome analysis. METHODS: Differences in protein expression between primary CRC with LNM (LNM CRC) and without LNM (non-LNM CRC) were assessed using methyl esterification stable isotope labeling coupled with 2D liquid chromatography followed by tandem mass spectrometry (2D-LC-MS/MS). The relationship to clinicopathological parameters and prognosis of candidate biomarkers was examined using an independent sample set. RESULTS: Forty-three proteins were found to be differentially expressed by at least 2.5-fold in two types of CRC. S100A4 was significantly upregulated in LNM CRC compared with non-LNM CRC, which was confirmed by Western blotting, immunohistochemistry and real-time quantitative polymerase chain reaction. Further immunohistochemistry on another 112 CRC cases showed that overexpression of S100A4 frequently existed in LNM CRC compared with non-LNM CRC (P<0.001). Overexpression of S100A4 was significantly associated with LNM (P<0.001), advanced TNM stage (P<0.001), increased 5-year recurrence rate (P<0.001) and decreased 5-year overall survival rate (P<0.001). Univariate and multivariate analyses indicated that S100A4 expression was an independent prognostic factor for recurrence and survival of CRC patients (P<0.05). CONCLUSION: S100A4 might serve as a powerful biomarker for LNM and a prognostic factor in CRC.

BACKGROUND AND OBJECTIVES: The calcium-binding protein S100A4 promotes tumor progression and metastasis. We investigated the expression of S100A4 in colorectal cancer and its clinicopathologic significance in colorectal cancer. METHODS: A total of 526 colorectal cancer patients were examined for S100A4 protein by immunohistochemistry using tissue microarrays. S100A4 mRNA was subsequently investigated by in situ hybridization. RESULTS: S100A4 protein was expressed in various cell types including tumor cells, but S100A4 mRNA was only expressed in tumor cells. Cytoplasmic expression of S100A4 protein was seen in 127 (24.1%) of 526 tumors and significantly correlated with older age, depth of invasion, lymph node metastasis, and worse overall survival. Nuclear expression of S100A4 protein was observed in 136 (25.9%) tumors and significantly related to the depth of invasion, perineural invasion, and worse overall survival. However, there was no correlation between S100A4 mRNA expression and clinicopathological parameters. Upon multivariate analysis nuclear expression of S100A4 protein was found to be an independent prognostic factor of poor survival. CONCLUSIONS: expression of S100A4 protein in colorectal cancers may indicate tumor progression and lymph node metastasis and can be useful for prediction of overall survival of patients with colorectal cancers.

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-inducing protein S100A4 was found to be a prognostic indicator for the development of metachronous metastases. S100A4 expression levels correlate with the formation of human colorectal cancer metastases and shorter patients survival. Inhibition of S100A4 expression in patients might therefore result in decreased metastasis formation and prolonged survival. In the present study, we used shRNA expression plasmids to inhibit S100A4 expression in the colorectal cancer cell lines HCT116, SW620 and DLD-1. Cell lines with reduced S100A4 expression showed reduced cell migration and invasion in vitro. The knock-down of S100A4 expression also led to significantly diminished formation of liver metastases when intrasplenically transplanted in mice (P = 0.004). We then focused on the therapeutic potential of systemically applied shRNA expression plasmids acting on S100A4 via repeated hydrodynamics-based tail vein injection of plasmid DNA. Mice, intrasplenically transplanted with HCT116 cells and treated systemically with S100A4shRNA plasmids, showed a decrease of S100A4 and MMP9 expression levels, resulting in significantly reduced liver metastases (P = 0.005). In summary, we show for the first time the intratumoral knock down of S100A4 via systemic application of S100A4shRNA plasmid DNA, which restricts metastasis formation in a xenografted mouse model of colorectal cancer.

OBJECTIVE: To study the role of S100A4 in the carcinogenesis, development, invasion and metastasis of esophageal squamous cell carcinoma. METHODS: Immunohistochemistry was used to detect the expressions of S100A4, MMP-2 and E-cadherin proteins in 100 cases of surgically resected esophageal squamous cell carcinoma specimens. RT-PCR and Western blot were used to detect the expressions of S100A4 mRNA and protein in esophageal squamous cell carcinoma line EC-1 and TE-1. Boyden-chamber model in vitro was utilized to detect the invasion ability of EC-1 and TE-1 cells. RESULTS: The positivity rate of S100A4 protein was 52.0% was in esophageal carcinoma tissues, significantly higher than that in normal tissues (26.0%) (P<0.01). The expression of S100A4 was related to tumor grading, invasive depth and lymph node metastasis (P<0.05). In esophageal carcinoma, the expression of S100A4 was positively correlated to MMP-2 expression (P<0.01), but inversely to E-cadherin expression (P<0.05). The expressions of S100A4 mRNA (0.894-/+0.021) and protein (0.897-/+0.053) in EC-1 cells were significantly higher than those in TE-1 (0.812-/+0.040 and 0.645-/+0.089, respectively, P<0.01), and the invasion ability of EC-1 cells was significantly higher than that of TE-1 cells (91.00-/+17.44 vs 61.80-/+11.10, P<0.01). CONCLUSION: The overexpression of S100A4 in esophageal squamous cell carcinoma tissue and highly invasive EC-1 cells may contribute to the carcinogenesis, development, invasion and metastasis of esophageal squamous cell carcinoma.

It is well documented that S100A4 is upregulated in a large amount of invasive tumors and plays a pivotal role in tumor invasion and metastasis. However, the precise role and mechanism S100A4 exerts in the invasion and metastasis of esophageal squamous cell carcinoma (ESCC) have not been fully elucidated to date. Our data demonstrated that S100A4 was overexpressed in human ESCC tissues, especially in ESCC with poor differentiation, deep invasion and lymph node metastasis. Subsequently, the knockdown of S100A4 by RNAi in ESCC cell line (EC-1) could reduce cell invasion, metastasis and proliferation ability in vitro. Most importantly, S100A4 regulated MMP-2 positively and E-cadherin negatively in vivo and in vitro to some extent. Our results suggest that S100A4 is an important factor in the invasion, metastasis and proliferation of ESCC and may control invasion and metastasis at least in part through the regulation of MMP-2 and E-cadherin activity. S100A4 may serve as a biomarker for progression of ESCC and a potential molecular target for biotherapy of ESCC.

OBJECTIVE: To investigate the intracellular localization of S100A4 in gastric carcinoma cells and the relationship between S100A4 expression status and lymph node metastasis of gastric carcinoma. METHODS: Western blotting analysis was performed to locate the expression of S100A4 protein in sub-fraction components of frozen tissues. S100A4 protein expression was also determined by immunohistochemical method in 131 samples of gastric cancer and 20 samples of matched metastatic lymph nodes. RESULTS: Thirty-two of 131 (24.4%) gastric carcinoma showed positive S100A4 nuclear expression and 50/131 (38.2%) carcinoma showed positive cytoplasmic expression. In 32 samples with positive S100A4 nuclear expression, 30 (93.8%) carcinomas had positive lymph node metastases. S100A4 nuclear expression level was higher in gastric carcinoma with lymph node metastasis (29.1%) than that without lymph node metastasis (7.1%) (P=0.016). CONCLUSION: Nuclear expression of S100A4 is associated with lymph node metastasis of gastric carcinoma.

BACKGROUND: The present study investigated the clinical significance of S100 calcium binding protein A4 in the development, progression, and metastasis of gastric cancer. METHODS: tumor tissue, adjacent normal tissue, and lymph node and peritoneal metastases were obtained from patients with gastric cancer, and their gene expression profiles were analyzed by Affymetrix GeneChip HG-U133A2.0 array. The expression of S100A4 was detected by real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) in gastric tumor tissue and lymph node and peritoneum metastasis. Immunohistochemistry was employed to analyze S100A4 expression in 436 clinicopathologically characterized gastric cancer cases and in corresponding distant metastases from 61 patients. RESULTS: A total of 434 genes and 169 expressed sequence tags were upregulated by at least twofold in the tumor tissue. The expression of S100A4 in lymph node and peritoneal metastases was significantly higher than that in gastric tumor tissue. The expression of S100A4 messenger RNA (mRNA) or protein differed significantly among gastric tumor tissue, matched normal gastric mucosa, and lymph node and peritoneal metastases. Further multivariate analysis suggested that depth of invasion, lymph node and distant metastases, tumor-node-metastasis (TNM) stage, and upregulation of S100A4 were independent prognostic indicators for the disease. CONCLUSION: Gene expression profiles are a useful way to perform simultaneously large-scale analysis of the expression level of thousands of genes. expression of S100A4 in gastric cancer is associated significantly with lymph node and distant metastases, and poor prognosis. S100A4 may be a useful marker to predict development, progression, and metastasis of gastric cancer.

Detachment from the extracellular matrix induces a form of programmed cell death termed anoikis. Resistance to anoikis permits cancer cells to survive in systemic circulation and facilitates their metastasis to distant organs. It is well known that S100A4 is overexpressed in many tumors and involved in tumor metastasis, but the mechanisms of the metastasis-promoting function of S100A4 are not fully understood. We hypothesized that S100A4 might play a role in anoikis of gastric cancer cells and further affects their metastasis. To test this hypothesis, we changed the expression of S100A4 by means of RNA interference or experimental overexpression and investigated the effect on anoikis. We found that knockdown of S100A4 by RNA interference led to significantly increased anoikis, whereas overexpression of S100A4 resulted in inhibition of anoikis. Furthermore, we provide evidence that inhibition of S100A4 resulted in the downregulation of alpha5 and alphav integrin expression. These findings suggest that S100A4 protects gastric cancer cells from anoikis by regulation of alphav and alpha5 integrin expression, which sheds a novel light for the role of S100A4 in cancer metastasis.

Overexpression of the S100 calcium-binding protein A4 (S100A4) is involved in epithelial-to-mesenchymal transition, oncogenic transformation, angiogenesis, cytoskeletal integrity and cancer metastasis. Here, we elucidated the role of S100A4 in tumorigenesis and progression of gastric carcinomas. S100A4 expression in gastric carcinomas, adenomas and adjacent non-neoplastic mucosa was analyzed by immunohistochemical, real-time reverse transcriptase (RT)-polymerase chain reaction (PCR) and western blot analyses, and was correlated with various clinicopathological parameters. S100A4 protein expression was increased gradually in the following order: gastritis (19.2%), intestinal metaplasia (IM; 23.3%), dysplasia (34.9%) and carcinoma (55.2%; P<0.001). S100A4 was positively correlated with tumor size, depth of invasion, lymphatic invasion, lymph node metastasis and tumor-node-metastasis (TNM) staging (P<0.05), but not with the age and gender of the carcinoma patients (P>0.05). Intestinal-type (IT) carcinomas showed a higher S100A4 expression than diffuse-type (DT) carcinomas (P<0.001). S100A4 mRNA expression also increased in the following order: gastritis < IM < dysplasia < carcinoma (P<0.05). S100A4 overexpression was observed in gastric carcinomas with a larger diameter, deeper invasion, lymph node metastasis and in IT carcinoma (P<0.05). Univariate analysis using the Kaplan-Meier method indicated a lower cumulative survival rate for patients with weak or moderate S100A4 expression compared with patients not expressing S100A4 (P<0.001). Multivariate analysis using Cox's proportional hazard model demonstrated that depth of invasion, lymphatic or venous invasion, lymph node metastasis, TNM staging and S100A4 expression were independent factors for poor patient prognosis (P<0.05). In conclusion, S100A4 upregulation is positively associated with the pathogenesis, growth, invasion, metastasis and differentiation of gastric carcinomas. S100A4 may be a promising marker indicative of the aggressive behavior and prognosis of gastric carcinomas.

The presence of cervical metastasis is responsible for high morbidity and mortality rates in individuals with head and neck squamous cell carcinoma (HNSCC). S100A4, a pleiotropic EF-hand calcium-binding protein, is expressed in various normal and cancer cell types. During cancer progression, molecular disturbances in S100A4 can modulate the activity and expression of pre-metastatic and metastatic genes. In this study, we investigated the association between S100A4 methylation status and protein expression as well as the expression of the S100A4 related-proteins annexin A2 (ANXA2), matrix metallopeptidase-9, and endoglin, for metastasis and other clinicopathological parameters in HNSCC. Formalin-fixed, paraffin-embedded blocks of metastatic and non-metastatic HNSCC and matched cervical lymph node (LN) samples (metastatic LN = mLN, non-metastatic = nmLN, and control LN (lymphadenitis) = cLN) were submitted for methylation specific-polymerase chain reaction and immunohistochemistry. Our results showed that S100A4 methylation status failed to demonstrate association with cervical metastasis and other clinicopathological factors related to HNSCC. HNSCC samples from patients that presented with metastatic disease showed high S100A4 and endoglin expression (p < 0.05). In conclusion, molecular disturbances in S100A4 and endoglin expression might regulate the formation of cervical metastasis in HNSCC.

Widespread metastasis of hepatocellular carcinoma (HCC) was a complex cascade of events, which is still beyond full appreciation. Screening key proteins, which play a critical role in metastasis, using high-throughput proteomics approach help discover valuable biomarkers and elucidate the mechanism of metastasis. This study was to find out some metastasis candidate proteins among HCC cell lines with various metastatic potential by comparative proteomics, and then further validate the biological function of these proteins in metastasis in vitro. The protein profiles of metastatic HCC cell lines (MHCC97H and MHCC97L) displayed obvious differences compared with nonmetastatic ones (Hep3B). Twenty-six metastasis candidate proteins, which were identified by on-line LC-ESI-MS/MS, such as S100 calcium-binding protein A4 (S100A4), annexin 1, etc., might have much application in diagnostic procedures and prognosis evaluation. S100A4, as a leading different metastasis candidate protein, which overexpressed only in the metastatic cells, was selected for further investigation. A series of assays related to invasion and metastasis in vitro, including cell motility, invasion, and matrix metalloproteinases (MMPs) secretion, were performed in MHCC97H/antisense recombinant plasmid to S100A4 (pcDNA3.1(+) AS S100A4) and the mock controls. All the data in the present study suggested that S100A4 might contribute to HCC invasion and metastasis through two paths of matrix metalloproteinase (MMP9) secretion regulation and strengthened motility and invasion properties.

BACKGROUND AND OBJECTIVES: We previously showed that the calcium-binding protein S100A4 is overexpressed and related to metastasis in hepatocellular carcinoma (HCC). However, whether S100A4 participates in the regulation of metastasis and its mechanisms in HCC is mostly unknown. Given the associations of S100A4, nuclear factor-kB (NF-kB/RelA) and MMP-9 with metastasis in a variety of malignancies, we explored a potential role of S100A4 in HCC metastasis and its mechanism. METHODS: 20 patients with HCC invasion (Lymph node metastasis, microvascular invasion, major portal vein invasion and intrahepatic metastasis) and 20 patients without HCC invasion were included. These tissues were detected for the expression of S100A4, NF-kB/RelA and MMP-9 by immunohistochemistry and quantitative real time polymerase chain reaction (Q-PCR). Correlation between the expressions of S100A4, NF-kB/RelA and MMP-9 with the invasion was analysed. The expressions of S100A4, nuclear factor-kB and MMP-9 was evaluated in HepG2 cells by western blot and immunohistochemistry. HepG2 cells were stably transfected with S100A4-specific small interfering RNA (S100A4 siRNA) to knockdown of S100A4, then transiently transfected with S100A4 cDNA to rescure the S100A4 level and evaluated for effects on invasion and expression analysis for molecules involved in invasion. After the HepG2 cells recurred the S100A4 levels, the HepG2 cells was treated with 5 microM Pyrrolidine Dithiocarbamate (PDTC) (a selective NF kappa B inhibitor) to inhibit the NF-kB activity, or treated with Batimast (BB94: a MMPs inhibitor) to inhibit the MMP-9 activity. The expression analysis for molecules involved in invasion was analyzed. RESULTS: A significant increase of S100A4, NF-kB/RelA and MMP-9 expression in HCC tissues with invasion than that of without invasion. A positive correlation was observed between S100A4, NF-kB/RelA, MMP-9 and invasion, respectively. In addition, S100A4 was positively correlated with NF-kB and MMP-9. S100A4 siRNA mediated knockdown of S100A4 in HepG2 cells resulted in significant reduction in the NF-kB activity and MMP-9 expression, and dramatically decreased its invasion. Moreover, the HepG2 cell metastatic potential was rescued by overexpression of S100A4 completely, at the same time, the NF-kB activity and MMP-9 expression was also increased. Pretreatment with PDTC or BB94 was observed to significantly reduce NF-kB activity and MMP-9 expression and dramatically decreased S100A4 -induced invasion. CONCLUSIONS: Our findings indicate that S100A4 contributes to HCC metastasis by activation of NF-kB dependent MMP-9 expression, suggesting S100A4 as a novel diagnostic biomarker and therapeutic target in HCC.

A loss or reduced expression of E-cadherin, the main cell-to-cell adhesion molecule, correlates with distant metastasis in various cancers. Recent studies have reported a close correlation between the expression of E-cadherin and that of S100A4, calcium-binding protein. In this study, we investigated the expression of E-cadherin and S100A4 status in relation to the clinicopathological parameters of pulmonary adenocarcinoma. We finely and quantitatively examined the expression of E-cadherin and S100A4 using real-time polymerase chain reaction (PCR) in a total of 92 pulmonary adenocarcinomas obtained by surgical resection. All of the pulmonary adenocarcinomas showed significant expression of E-cadherin and S100A4. Real-time PCR showed lower E-cadherin expression in 21 adenocarcinomas, while 71 adenocarcinomas expressed a higher expression of E-cadherin. Of 21 adenocarcinomas with lower-expressing E-cadherin, 12 showed a higher expression of S100A4. These 12 cases significantly showed a poorer prognosis than others (p=0.047, Kaplan-Meier, log-rank test) and significantly showed more frequent venous involvement than others (p=0.042, chi2 test). These results suggested that reduced E-cadherin expression combined with higher S100A4 expression is related to a poor prognosis through hematogenous metastasis in pulmonary adenocarcinoma.

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.

The present study is aimed at studying the gene for TIMP-3, a mammalian tissue inhibitor, by constructing a recombinant eukaryotic cell vector for gene therapy in human breast cancer. We obtained the TIMP-3 gene from the human placent by RT-PCR. TIMP-3 gene was subcloned into pcDNA3.1 vetor from pMD18T vector by means of gene cloning to construct pcDNA3.1 recombinant vector. Human breast cancer cell line MDA-MB-453 was transfected with pcDNA3.1-TIMP3 recombinant vector using lipofectamine reagent. Then the expression of TIMP-3 and the effect on the metastasis of MDA-MB-453 were examined. The correct construction of pcDNA-TIMP3 was identified by means of restriction enzyme analysis, PCR amplication and nucleotide sequencing. Western blotting showed that the transfected cells were able to express TIMP-3, indicating that our construction of the pcDNA-TIMP3 eukaryotic expression vector was constructed successfully. Our experiments further indicated that the potential of metastasis was significantly reduced for the transfected cell line MDA-MB-453.

A loss or reduced expression of E-cadherin, the main cell-to-cell adhesion molecule, correlates with distant metastasis in various cancers. Recent studies have reported a close correlation between the expression of E-cadherin and that of S100A4, calcium-binding protein. In this study, we investigated the expression of E-cadherin and S100A4 status in relation to the clinicopathological parameters of pulmonary adenocarcinoma. We finely and quantitatively examined the expression of E-cadherin and S100A4 using real-time polymerase chain reaction (PCR) in a total of 92 pulmonary adenocarcinomas obtained by surgical resection. All of the pulmonary adenocarcinomas showed significant expression of E-cadherin and S100A4. Real-time PCR showed lower E-cadherin expression in 21 adenocarcinomas, while 71 adenocarcinomas expressed a higher expression of E-cadherin. Of 21 adenocarcinomas with lower-expressing E-cadherin, 12 showed a higher expression of S100A4. These 12 cases significantly showed a poorer prognosis than others (p=0.047, Kaplan-Meier, log-rank test) and significantly showed more frequent venous involvement than others (p=0.042, chi2 test). These results suggested that reduced E-cadherin expression combined with higher S100A4 expression is related to a poor prognosis through hematogenous metastasis in pulmonary adenocarcinoma.

The metastasis-promoting protein S100A4 stimulates metastatic progression through both intracellular and extracellular functions. Extracellular activities of S100A4 include stimulation of angiogenesis, regulation of cell death and increased cell motility and invasion, but the exact molecular mechanisms by which extracellular S100A4 exerts these effects are incompletely elucidated. The aim of the present study was to characterize S100A4-induced signal transduction mechanisms and to identify S100A4 target genes. We demonstrate that extracellular S100A4 activates the transcription factor NF-kappaB in a subset of human cancer cell lines through induction of phosphorylation and subsequent degradation of the NF-kappaB inhibitor IkappaBalpha. Concomitantly, S100A4 induced a sustained activation of the MAP kinase JNK, whereas no increased activity of the MAP kinases p38 or ERK was observed. Microarray analyses identified 136 genes as being significantly regulated by S100A4 treatment, and potentially interesting S100A4-induced gene products include IkappaBalpha, p53, ephrin-A1 and optineurin. Increased expression of ephrin-A1 and optineurin was validated using RT-PCR, Western blotting and functional assays. Furthermore, S100A4-stimulated transcription of these target genes was dependent on activation of the NF-kappaB pathway. In conclusion, these findings contribute to the understanding of the complex molecular mechanisms responsible for the diverse biological functions of extracellular S100A4, and provide further evidence of how S100A4 may stimulate metastatic progression.

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.

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 calcium-binding protein S100A4 promotes metastasis in several experimental animal models, and S100A4 protein expression is associated with patient outcome in a number of tumor types. S100A4 is localized in the nucleus, cytoplasm, and extracellular space and possesses a wide range of biological functions, such as regulation of angiogenesis, cell survival, motility, and invasion. In this review, we summarize the evidence connecting S100A4 and cancer metastasis and discuss the mechanisms by which S100A4 promotes tumor progression.

PCR and direct DNA sequencing methods were used to analyse the prevalence of mutations in exon 2 of the p21waf1 gene in 14 oral squamous cell carcinomas (OSCCs) and 8 non-malignant oral mucosal lesions from Sudanese toombak dippers. For comparison, OSCCs (14 from the Sudan, 16 from Norway, 11 from Sweden, 21 from the USA and 14 from the UK) and non-malignant oral mucosal lesions (3 from the Sudan) from non-snuff-dippers were included. The prevalence of mutations in exons 2 & 3 of the S100A4 gene were analysed in the 14 OSCCs from toombak-dippers and in 25 cases of OSCCs from the control non-snuff-dippers. Of the 14 OSCCs investigated from toombak-dippers, mutations in the p21waf1 exon 2 were found in 43% (6 out of 14), compared to 14% (2 out of 14), 22% (6 out of 27) and 14% (5 out of 35) found in those from non-snuff-dippers from the Sudan, Scandinavia and the USA/UK, respectively. OSCCs from toombak-dippers showed 13 different mutations distributed as 10 (77%) transitions and 3 (23%) transversions. OSCCs from non-snuff-dippers from the Sudan, Scandinavia, the USA and the UK showed 33 different mutations distributed as 14 (42%) transitions and 19 (58%) transversions. In the OSCCs examined, cases with mutations in the p21waf1 also had p53 gene mutations. Only exon 2 of the S100A4 gene was found mutated in 3 cases of OSCCs (one from a toombak-dipper and two from the non-snuff-dippers). The toombak-dipper OSCC had 4 mutations (one transition, 3 transversions), compared to the OSCCs from non-snuff-dippers which showed 3 mutations each (one transition, 2 transversions). All these 3 cases were negative for mutations in the p21waf1 and p53 genes. No mutations of p21waf1 or S100A4 were found in the non-malignant oral mucosal lesions from the snuff-dippers/non-dippers. These findings suggest that; (i) p21waf1, together with p53, is a target gene of oral carcinogenesis in OSCCs from toombak-dippers, with the tobacco specific nitrosamines present in toombak possibly acting as principal carcinogens in these OSCCs; (ii) findings of p21waf1 exon 2 mutations in the OSCCs unrelated to snuff use further demonstrate that this gene may play an important role during the pathogenesis of OSCCs caused by smoked tobacco use; (iii) mutations in the S100A4 gene are rare in OSCCs, but appears to be complementary to p21waf1 and p53 mutations. Since molecular analysis of OSCCs can provide clues to endogenous or environmental factors contributing to the high risk of OSCCs, further analysis of the role of the p21waf1 gene mutations as a biomarker of malignant transformation, which is linked to the p53 gene, is necessary, especially in habitual users of toombak from the Sudan.

To study the role of the metastasis associated protein S100A4, an osteosarcoma cell line (OHS) with a high level of this protein was transfected with a vector containing a ribozyme that degrades S100A4 mRNA and, as controls, OHS cells were transfected with the vector alone. We have followed up our previous investigation (Bjornland et al. 1999) by a detailed investigation of these cell lines' synthesis of MMP and TIMP proteins at different cell densities. It is shown that the cell lines with a low S100A4 level produced a reduced amount of immunoreactive MMP-2 at cellular subconfluence, while at confluence there was no difference compared to the control cells. The cell lines with a reduced S100A4 level produced less of the activated form of MMP-2 (62-kDa) and less TIMP-1 than the corresponding control cells, independent of cell density. Isolated cell membranes from cell lines with a reduced S100A4 level contained less MT1-MMP, MMP-2 and TIMP-2 compared to the control cells. Activation of exogenously added proMMP-2 was less effective with the former membrane preparations. It appeared that the mechanism behind the S100A4 dependent activation of proMMP-2 varied with cell density, as SN50, a peptide inhibitor of NF-kappaB nuclear translocation reduced the activation of MMP-2 at low cell density, but had no effect at high cell density. Thus, one of the mechanisms by which S100A4 may exert its effect on metastasis of some tumors is by regulating the MMP-2 activity.

Osteosarcoma is a rare but aggressive bone neoplasm in humans, which is commonly treated with surgery, classical chemotherapy and radiation. Sorafenib, an inhibitor of a number of kinases targeting the Raf/MEK/ERK pathway, is a promising new chemotherapeutic agent in human medicine that has been approved since 2006 for the therapy of renal cell carcinoma and since 2007 for the treatment of hepatocellular carcinoma. Here, we studied the antimetastatic potential of 4 microM of this multikinase inhibitor in a human osteosarcoma cell line. DNA microarray-based gene expression profiling detected 297 and 232 genes upregulated or downregulated at a threshold of >2-fold expression alteration (P<0.05) in the sorafenib-treated cells. Three genes (CXCR4, FOS and S100A4) that are involved in tumor progression were chosen for validation by quantitative PCR (qPCR) and protein expression analysis. The decrease in RNA expression detected by microarray profiling was confirmed by qPCR for all three genes (P<0.01). On the protein level, sorafenib-induced reduction of S100A4 was verified both by western blotting and immunohistochemistry. For CXCR4 and c-Fos, a reduced protein expression was shown by immunohistochemistry, for c-Fos also by immunoblotting. We conclude that sorafenib could serve as a potent chemotherapeutical agent by which to inhibit the metastatic progression of osteosarcomas.

The calcium-binding protein, S100A4, with an inverse association of E-cadherin, is known to correlate with prognosis in various cancers. In this study, we investigated the expression of the S100A4 and E-cadherin status in relation to the clinicopathological parameters of pancreatic cancer. The expression status of these two proteins was examined in 72 specimens of primary pancreatic carcinoma with immunohistochemistry. Fifty-six of 72 (78%) surgical specimens of primary pancreatic cancer were positive for S100A4 according to immunohistochemistry. Thirty-one (43%) specimens of pancreatic cancer showed positive expression of E-cadherin. The inverse association of S100A4 and E-cadherin expression was significant in the cancers (p < 0.0001). The S100A4 expression correlated significantly with the pathological T stage and poorer prognosis (p = 0.024). The 41 E-cadherin-negative cases showed poorer prognoses and a higher incidence of liver metastasis (p = 0.0344, p = 0.027). The 10 cases with S100A4-negative/E-cadherin-positive cancers showed a significantly better prognosis than the others (p < 0.05). The histological grade (p = 0.004), nodal status (p < 0.0001) and S100A4-positive status (p = 0.048) were highly significant independent prognostic predictors (p < 0.05). These results suggest that S100A4 overexpression combined with reduced E-cadherin expression play important roles in tumor progression and metastasis in pancreatic cancer. The combined examination of these two molecules is useful in evaluating the outcome of pancreatic cancer patient.

We previously showed that the calcium-binding protein S100A4 is overexpressed during the progression of prostate cancer (CaP) in humans and in the TRAMP (transgenic adenocarcinoma of the mouse prostate) mouse model. We tested a hypothesis that the S100A4 gene plays a role in the invasiveness of human CaP and may be associated with its metastatic spread. We observed that siRNA-mediated suppression of the S100A4 gene significantly reduced the proliferative and invasive capability of the highly invasive CaP cells PC-3. We evaluated the mechanism through which the S100A4 gene controls invasiveness of cells by using a macroarray containing 96 well characterized metastatic genes. We found that matrix metalloproteinase 9 (MMP-9) and its tissue inhibitor (TIMP-1) were highly responsive to S100A4 gene suppression. Furthermore, S100A4 suppression significantly reduced the expression and proteolytic activity of MMP-9. By employing an MMP-9-promoter reporter, we observed a significant reduction in the transcriptional activation of the MMP-9 gene in S100A4-siRNA-transfected cells. Cells overexpressing the S100A4 gene (when transfected with pcDNA3.1-S100A4 plasmid) also significantly expressed MMP-9 and TIMP-1 genes with increased proteolytic activity of MMP-9 concomitant to increased transcriptional activation of the MMP-9 gene. S100A4-siRNA-transfected cells exhibited a reduced rate of tumor growth under in vivo conditions. Our data demonstrate that the S100A4 gene controls the invasive potential of human CaP cells through regulation of MMP-9 and that this association may contribute to metastasis of CaP cells. We suggest that S100A4 could be used as a biomarker for CaP progression and a novel therapeutic or chemopreventive target for human CaP treatment.

tumor cell invasion and metastasis are the hallmark of malignant neoplasm. Despite advances in the management of thyroid carcinoma and other solid tumors, metastasis continues to be the most significant cause in cancer mortality. To gain new insights into this complex process in thyroid carcinoma, we established a thyroid carcinoma cell line (ARO-met2) with high metastatic capacity to the lung by sequential passage of a human anaplastic thyroid cancer cell line (ARO) through the lung of a nude mouse. Global patterns of gene expression were analyzed in cells of the parental ARO and the ARO-met2, using Atlas human cancer 1.2 array with 1176 cancer-related genes. In total, 184 genes were differentially expressed more than 1.5 times, and 64 genes were differentially expressed over two times. Among those 64 genes, 43 were overexpressed, and 21 genes were underexpressed. Many genes whose increased expression was thought to be related to tumor progression were identified, such as c-Met, ezrin, integrin, motility-related protein-1, cadherin, and S100A4. The most highly expressed gene is the S100A4 (8-fold higher than control), which is a member of a small calcium binding protein family and is involved in the cell proliferation and cancer progression. The S100A4 overexpression in the ARO-met2 cells was later confirmed by Northern blot and real-time reverse transcriptase-PCR. Analysis of 49 thyroid tumor specimens by real-time reverse transcriptase-PCR (eight benign goiters, 36 papillary, and five anaplastic carcinomas) revealed that S100A4 overexpression was present in most advanced thyroid carcinomas and lymph node metastases, and was associated with poor prognosis. None of the benign goiters was found to have S100A4 overexpression. These data suggest that S100A4 could be used as a prognostic marker for thyroid carcinoma. Given that S100A4 is involved in tumor progression and metastasis, it may be a potential target for therapeutic intervention.

tumour cell invasion and metastasis are the hallmark of malignant neoplasm. S100A4 is a member of small calcium-binding protein family and is involved in the cell proliferation and cancer progression. S100A4 is capable of inducing metastasis in animal models and is associated with aggressive phenotype of human tumours. We previously identified S100A4 as a candidate gene involved in anaplastic thyroid cancer metastasis by microarray analysis. To further determine whether S100A4 overexpression is associated with thyroid tumour invasion and metastasis, in the present study, we examined S100A4 gene expression in six benign multinodular goitres (MNG) and 28 matched samples of adjacent normal thyroid tissue (N), primary (T) and metastatic (M) papillary thyroid carcinomas (PTC) by immunohistochemistry and real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis. This gave us the advantage of directly comparing levels of S100A4 expression within the same genetic background. Using immunohistochemistry, we found that high levels of S100A4 were detected in 24 of 28 (86%) PTC specimens and their local regional lymph node or distant metastases. No S100A4 staining was observed in normal thyroid tissues and simple MNG. However, in MNG coexistent with PTC, moderate focal staining could be found in 11 of 15 MNG adjacent to PTC. The S100A4 was stained more intensely in invading fronts than in central portions of both T and M. Real-time RT-PCR analysis of primary tumours and their matched lymph node metastasis demonstrated that significantly higher S100A4 transcripts were present in metastatic tumours as compared to the primary tumours (P<0.01). These data suggest that overexpression of S100A4 is associated with thyroid tumour invasion and metastasis and it may be a potential target for therapeutic intervention.