F806 Suppresses the Invasion and Metastasis of Esophageal Squamous Cell Carcinoma via Downregulating F-Actin Assembly-Related Rho Family Proteins.

Invasion and metastasis are critical pathological and mortal processes in esophageal squamous cell carcinoma (ESCC). Novel drugs, targeting the two cancer migration stages, will augment the treatment options for ESCC therapy and improve overall survival. A novel natural macrolide F806 specifically promotes apoptosis of various ESCC cells. However, whether F806 can inhibit metastasis of ESCC cells needs further evaluation. Here, our data showed that F806 inhibits dynamic F-actin assembly and then suppresses the migration of ESCC cells in vitro and their invasion and metastasis in vivo. The correlation between cancer migration and actin cytoskeleton assembly was consistent with the ability of F806 to prevent the aggregation of Paxillin, an essential protein for focal adhesion formation through binding to the ends of actin filaments. Furthermore, F806 downregulated the expression and activity of the Rho family proteins cell division cycle 42 (CDC42), RAC family small GTPase 1 (RAC1), and RAS homolog family member A (RHOA). Taken together, these results suggest that F806 can suppress cancer invasion and metastasis via interrupting the assembly of migration components involving F-actin.

Riboflavin Depletion Promotes Tumorigenesis in HEK293T and NIH3T3 Cells by Sustaining Cell Proliferation and Regulating Cell Cycle-Related Gene Transcription.

Background: Riboflavin is an essential component of the human diet and its derivative cofactors play an established role in oxidative metabolism. Riboflavin deficiency has been linked with various human diseases. Objective: The objective of this study was to identify whether riboflavin depletion promotes tumorigenesis. Methods: HEK293T and NIH3T3 cells were cultured in riboflavin-deficient or riboflavin-sufficient medium and passaged every 48 h. Cells were collected every 5 generations and plate colony formation assays were performed to observe cell proliferation. Subcutaneous tumorigenicity assays in NU/NU mice were used to observe tumorigenicity of riboflavin-depleted HEK293T cells. Mechanistically, gene expression profiling and gene ontology analysis were used to identify abnormally expressed genes induced by riboflavin depletion. Western blot analyses, cell cycle analyses, and chromatin immunoprecipitation were used to validate the expression of cell cycle-related genes. Results: Plate colony formation of NIH3T3 and HEK293T cell lines was enhanced >2-fold when cultured in riboflavin-deficient medium for 10-20 generations. Moreover, we observed enhanced subcutaneous tumorigenicity in NU/NU mice following injection of riboflavin-depleted compared with normal HEK293T cells (55.6% compared with 0.0% tumor formation, respectively). Gene expression profiling and gene ontology analysis revealed that riboflavin depletion induced the expression of cell cycle-related genes. Validation experiments also found that riboflavin depletion decreased p21 and p27 protein levels by ∼20%, and increased cell cycle-related and expression-elevated protein in tumor (CREPT) protein expression >2-fold, resulting in cyclin D1 and CDK4 levels being increased ∼1.5-fold, and cell cycle acceleration. We also observed that riboflavin depletion decreased intracellular riboflavin levels by 20% and upregulated expression of riboflavin transporter genes, particularly SLC52A3, and that the changes in CREPT and SLC52A3 correlated with specific epigenetic changes in their promoters in riboflavin-depleted HEK293T cells. Conclusion: Riboflavin depletion contributes to HEK293T and NIH3T3 cell tumorigenesis and may be a risk factor for tumor development.

The interaction of lncRNA EZR-AS1 with SMYD3 maintains overexpression of EZR in ESCC cells.

EZR, a member of the ezrin-radixin-moesin (ERM) family, is involved in multiple aspects of cell migration and cancer. SMYD3, a histone H3-lysine 4 (H3-K4)-specific methyltransferase, regulates EZR gene transcription, but the molecular mechanisms of epigenetic regulation remain ill-defined. Here, we show that antisense lncRNA EZR-AS1 was positively correlated with EZR expression in both human esophageal squamous cell carcinoma (ESCC) tissues and cell lines. Both in vivo and in vitro studies revealed that EZR-AS1 promoted cell migration through up-regulation of EZR expression. Mechanistically, antisense lncRNA EZR-AS1 formed a complex with RNA polymerase II to activate the transcription of EZR. Moreover, EZR-AS1 could recruit SMYD3 to a binding site, present in a GC-rich region downstream of the EZR promoter, causing the binding of SMYD3 and local enrichment of H3K4me3. Finally, the interaction of EZR-AS1 with SMYD3 further enhanced EZR transcription and expression. Our findings suggest that antisense lncRNA EZR-AS1, as a member of an RNA polymerase complex and through enhanced SMYD3-dependent H3K4 methylation, plays an important role in enhancing transcription of the EZR gene to promote the mobility and invasiveness of human cancer cells.

L1CAM drives oncogenicity in esophageal squamous cell carcinoma by stimulation of ezrin transcription.

L1 cell adhesion molecule (L1CAM) is highly expressed in various types of human cancers, displaying yet unknown molecular mechanisms underlying their oncogenic potential. Here, we found that L1CAM expression was significantly increased in esophageal squamous cell carcinoma (ESCC; n = 157) lesions compared with non-cancerous tissues. High tumorous L1CAM expression significantly correlated with reduced overall survival. Experimentally, L1CAM knockdown led to decreased cell growth, migration, and invasiveness in vitro, whereas overexpression of L1CAM showed the opposite effect. In nude mice, L1CAM depletion attenuated tumorigenesis and ability to penetrate the tissues surrounding ESCC cells. Gene set enrichment analysis (GSEA) and SubpathwayMiner analysis on gene expression profiles (microarray data on ESCC tissues, GSE53625; cDNA microarray data on L1CAM-knockdown ESCC cell line, GSE86268) suggested that L1CAM-co-expression genes were related to cell motility, cell proliferation, and regulation of actin cytoskeleton, validating the above experimental findings. Further mechanistical analysis showed that L1CAM upregulated the expression of the cytoskeletal protein ezrin via activating integrin ß1/MAPK/ERK/AP1 signaling and thus led to the malignant phenotypes of ESCC cells. Together, our findings suggest that L1CAM may be employed as a valuable prognosis marker and a therapeutic target for ESCC patients and that L1CAM promotes ESCC tumorigenicity by upregulating ezrin expression. KEY MESSAGES: L1CAM promotes growth and invasiveness of ESCC cells in vitro and in vivo. L1CAM upregulates the expression of ezrin by integrin α5ß1/MAPK/ERK/AP1 pathway. Ezrin is a key downstream effector in the L1CAM-promoted malignant phenotypes. High expression levels of both L1CAM and ezrin significantly correlated with reduced overall survival. Nuclear L1CAM is an independent prognosis marker for esophageal squamous cell carcinoma.

Ezrin Ser66 phosphorylation regulates invasion and metastasis of esophageal squamous cell carcinoma cells by mediating filopodia formation.

BACKGROUND: Ezrin, links the plasma membrane to the actin cytoskeleton, and plays an important role in the development and progression of human esophageal squamous cell carcinoma (ESCC). However, the roles of ezrin S66 phosphorylation in tumorigenesis of ESCC remain unclear. METHODS: Distribution of ezrin in membrane and cytosol fractions was examined by analysis of detergent-soluble/-insoluble fractions and cytosol/membrane fractionation. Both immunofluorescence and live imaging were used to explore the role of ezrin S66 phosphorylation in the behavior of ezrin and actin in cell filopodia. Cell proliferation, migration and invasion of ESCC cells were investigated by proliferation and migration assays, respectively. Tumorigenesis, local invasion and metastasis were assessed in a nude mouse model of regional lymph node metastasis. RESULTS: Ezrin S66 phosphorylation enhanced the recruitment of ezrin to the membrane in ESCC cells. Additionally, non-phosphorylatable ezrin (S66A) significantly prevented filopodia formation, as well as caused a reduction in the number, length and lifetime of filopodia. Moreover, functional experiments revealed that expression of non-phosphorylatable ezrin (S66A) markedly suppressed migration and invasion but not proliferation of ESCC cells in vitro, and attenuated local invasion and regional lymph node metastasis, but not primary tumor growth of ESCC cells in vivo. CONCLUSION: Ezrin S66 phosphorylation enhances filopodia formation, contributing to the regulation of invasion and metastasis of esophageal squamous cell carcinoma cells.

Biological characterization of three immortalized esophageal epithelial cell lines.

The key molecular events that contribute to tumorigenesis are incompletely understood. The aim of the present study was to characterize and compare the biological phenotypes of three human telomerase reverse transcriptase (hTERT) and/or human papillomavirus 16 E6 and E7­immortalized esophageal epithelial cell lines, NE2­hTERT (NE2), NE3­E6E7­hTERT (NE3) and NEcA6­E6E7­hTERT (NEcA6). The present study used soft­agar colony formation assays, tumorigenicity assays in nude mice, and cell proliferation, adhesion and migration assays to identify the biological characteristics of NE2, NE3 and NEcA6 cells. NE2 and NE3 cells exhibited characteristics of benign cells, such as the inability to grow in soft agar or form tumors in nude mice. By contrast, NEcA6 cells had undergone transformation, as demonstrated by the ability to grow in soft agar and form tumors in nude mice. In addition, NEcA6 cells exhibited increased migration and adhesion capabilities when compared with NE2 and NE3 cells. In order to identify mechanism(s) that may contribute to the altered biological phenotypes exhibited by these cells, the expression of three proteins involved in modulating cell migration [fascin, ezrin/radixin/moesin family proteins and phosphorylated­focal adhesion kinase (Tyr 397)], as well as the expression status and subcellular localization of three key focal adhesions components (paxillin, talin and kindlin­2) were examined. Paxillin, talin and kindlin­2 were localized to adhesive sites that connect F­actin with the extracellular matrix in transformed NEcA6 cells, but were distributed in a diffuse manner in NE2 and NE3 cells. Knockdown of kindlin­2 in NE3 and NEcA6 cells decreased cell adhesion, however, NEcA6 cells demonstrated a greater sensitivity to knockdown of kindlin­2. No significant differences were observed in the protein expression levels of fascin, exrin/radixin/moesin and p­FAK in the three cell lines. In conclusion, these results demonstrate that these three focal adhesion components, particularly kindlin­2, may contribute to the carcinogenesis of esophageal squamous cells.

SMYD3 stimulates EZR and LOXL2 transcription to enhance proliferation, migration, and invasion in esophageal squamous cell carcinoma.

Epigenetic alterations, including DNA methylation and histone modifications, are involved in the regulation of cancer initiation and progression. SET and MYND domain-containing protein 3 (SMYD3), a methyltransferase, plays an important role in transcriptional regulation during human cancer progression. However, SMYD3 expression and its function in esophageal squamous cell carcinoma (ESCC) remain unknown. In this study, SMYD3 expression was studied by immunohistochemistry in a tumor tissue microarray from 131 cases of ESCC patients. Statistical analysis showed that overall survival of patients with high SMYD3 expressing in primary tumors was significantly lower than that of patients with low SMYD3-expressing tumors (P = .008, log-rank test). Increased expression of SMYD3 was found to be associated with lymph node metastasis in ESCC (P = .036) and was an independent prognostic factor for poor overall survival (P = .025). RNAi-mediated knockdown of SMYD3 suppressed ESCC cell proliferation, migration, and invasion in vitro and inhibited local tumor invasion in vivo. SMYD3 regulated transcription of EZR and LOXL2 by directly binding to the sequences of the promoter regions of these target genes, as demonstrated by a chromatin immunoprecipitation assay. Immunohistochemical staining of ESCC tissues also confirmed that protein levels of EZR and LOXL2 positively correlated with SMYD3 expression, and the Spearman correlation coefficients (rs) were 0.78 (n = 81; P < .01) and 0.637 (n = 103; P < .01), respectively. These results indicate that SMYD3 enhances tumorigenicity in ESCC through enhancing transcription of genes involved in proliferation, migration, and invasion.

Lipocalin 2 promotes the migration and invasion of esophageal squamous cell carcinoma cells through a novel positive feedback loop.

Lipocalin 2 (LCN2) is a poor prognostic factor in esophageal squamous cell carcinoma (ESCC), however its functional roles and molecular mechanisms of action remain to be clarified. Here, we described the functions and signaling pathways for LCN2 in ESCC. Overexpression of LCN2 in ESCC cells accelerated cell migration and invasion in vitro, and promoted lung metastasis in vivo. Blocking LCN2 expression inhibited its pro-oncogenic effect. Either overexpression of LCN2 or treatment with recombinant human LCN2 protein enhanced the activation of MEK/ERK pathway, which in turn increases endogenous LCN2 to increase MMP-9 activity. The decreased p-cofilin and increased p-ERM induced by pERK1/2 cause the cytoskeleton F-actin rearrangement and alter the behavior of ESCC cells mediated by LCN2. As a consequence, activation of MMP-9 and the rearrangement of F-actin throw light on the mechanisms for LCN2 in ESCC. These results imply that LCN2 promotes the migration and invasion of ESCC cells through a novel positive feedback loop.

Macrolide analog F806 suppresses esophageal squamous cell carcinoma (ESCC) by blocking ß1 integrin activation.

The paucity of new drugs for the treatment of esophageal squamous cell carcinoma (ESCC) limits the treatment options. This study characterized the therapeutic efficacy and action mechanism of a novel natural macrolide compound F806 in human ESCC xenograft models and cell lines. F806 inhibited growth of ESCC, most importantly, it displayed fewer undesirable side effects on normal tissues in two human ESCC xenograft models. F806 inhibited proliferation of six ESCC cells lines, with the half maximal inhibitory concentration (IC50) ranging from 9.31 to 16.43 µM. Furthermore, F806 induced apoptosis of ESCC cells, contributing to its growth-inhibitory effect. Also, F806 inhibited cell adhesion resulting in anoikis. Mechanistic studies revealed that F806 inhibited the activation of ß1 integrin in part by binding to a novel site Arg610 of ß1 integrin, suppressed focal adhesion formation, decreased cell adhesion to extracellular matrix and eventually triggered apoptosis. We concluded that F806 would potentially be a well-tolerated anticancer drug by targeting ß1 integrin, resulting in anoikis in ESCC cells.

12-O-Tetradecanoylphorbol-13-Acetate Induces Up-Regulated Transcription of Variant 1 but Not Variant 2 of VIL2 in Esophageal Squamous Cell Carcinoma Cells via ERK1/2/AP-1/Sp1 Signaling.

The membrane-cytoskeleton link organizer ezrin may be the most "dramatic" tumor marker, being strongly over-expressed in nearly one-third of human malignancies. However, the molecular mechanisms of aberrant ezrin expression still need to be clarified. Ezrin, encoded by the VIL2 gene, has two transcript variants that differ in the transcriptional start site (TSS): V1 and V2. Both V1 and V2 encode the same protein. Here, we found that 12-O-tetradecanoylphorbol-13-acetate (TPA) induced over-expression of human VIL2 in esophageal squamous cell carcinoma (ESCC) cells. Furthermore, VIL2 V1 but not V2 was up-regulated after TPA stimulation in a time-dependent manner. AP-1 and Sp1 binding sites within the promoter region of VIL2 V1 acted not only as basal transcriptional elements but also as a composite TPA-responsive element (TRE) for the transcription of VIL2 V1. TPA stimulation enhanced c-Jun and Sp1 binding to the TRE via activation of the ERK1/2 pathway and increased protein levels of c-Jun, c-Fos, and Sp1, resulting in over-expression of VIL2 V1, whereas the MEK1/2 inhibitor U0126 blocked these events. Finally, we showed that TPA promoted the migration of ESCC cells whereas MEK1/2 inhibitor or ezrin silencing could partially inverse this alteration. Taken together, these results suggest that TPA is able to induce VIL2 V1 over-expression in ESCC cells by activating MEK/ERK1/2 signaling and increasing binding of Sp1 and c-Jun to the TRE of the VIL2 V1 promoter, and that VIL2 is an important TPA-induced effector.

Quantitative proteomics reveals the downregulation of GRB2 as a prominent node of F806-targeted cell proliferation network.

High-throughput proteomics has successfully identified thousands of proteins as potential therapeutic targets during investigations into mechanisms of drug action. A novel macrolide analog, denoted F806, is a potential antitumor drug. Here, using the quantitative proteomic approach of stable isotope labeling with amino acids in cell culture (SILAC) coupled to high-resolution mass spectrometry (MS), we characterize the F806-regulating protein profiles and identify the potential target molecules or pathways of F806 in esophageal squamous cell carcinoma (ESCC) cells. From a total of 1931 quantified proteins, 181 proteins were found to be down-regulated (FDR p-value<0.1, H/L ratio<0.738), and 119 proteins were up-regulated (FDR p-value<0.1, H/L ratio>1.156). Among the down-regulated proteins, we uncovered the over- and under-represented protein clusters in biological process and molecular function respectively by Gene Ontology analysis. Furthermore, down-regulated and up-regulated proteins were significantly enriched in 37 pathways and 60 sub-pathways by bioinformatic analysis (FDR p-value<0.1), while a down-regulated molecule growth factor receptor-bound protein 2 (GRB2) was a prominent node in fourteen cell proliferation-related sub-pathways. We concluded that GRB2 downregulation would be a potential target of F806 in ESCC cells. BIOLOGICAL SIGNIFICANCE: This study used SILAC-based quantitative proteomics screen to systematically characterize molecular changes induced by a novel macrolide analog F806 in esophageal squamous cell carcinoma (ESCC) cells. Followed by bioinformatic analyses, signal pathway networks generated from the quantified proteins, would facilitate future investigation into the further mechanisms of F806 in ESCC cells. Notably, it provided information that growth factor receptor-bound protein 2 (GRB2) would be a prominent node in the F806-targeted cell proliferation network.

ATF3 functions as a novel tumor suppressor with prognostic significance in esophageal squamous cell carcinoma.

ATF3 was a transcription factor involved in the progression of certain cancers. Here, we sought to explore the expression and biological function of ATF3 in esophageal squamous cell carcinomas (ESCC). The prognostic significance of ATF3 expression was evaluated in 150 ESCC samples and 21 normal squamous cell epithelium tissues. Results showed that ATF3 was down-regulated in ESCC lesions compared with paired non-cancerous tissues and low tumorous ATF3 expression significantly correlated with shorter overall survival (OS) and disease-free survival (DFS). Cox regression analysis confirmed that ATF3 expression was an independent prognostic factor. Experimentally, forced expression of ATF3 led to decreased growth and invasion properties of ESCC cells in vitro and in vivo, whereas knockdown of ATF3 did the opposite. Furthermore, ATF3 upregulated the expression of MDM2 by increasing the nuclear translocation of P53 and formed an ATF3/MDM2/MMP-2 complex that facilitated MMP-2 degradation, which subsequently led to inhibition of cell invasion. Finally, we showed that Cisplatin could restrain the invasion of ESCC cells by inducing the expression of ATF3 via P53 signaling. Combined, our findings highlight a suppressed role for ATF3 in ESCC and targeting ATF3 might be a potential therapeutic strategy.

Down-regulated desmocollin-2 promotes cell aggressiveness through redistributing adherens junctions and activating beta-catenin signalling in oesophageal squamous cell carcinoma.

In contrast to the well-recognized loss of adherens junctions in cancer progression, the role of desmosomal components in cancer development has not been well explored. We previously demonstrated that desmocollin-2 (DSC2), a desmosomal cadherin protein, is reduced in oesophageal squamous cell carcinoma (ESCC), and is associated with enhanced tumour metastasis and poor prognosis. Here, we report that restoration of DSC2 in ESCC cells impeded cell migration and invasion both in vitro and in vivo, whereas siRNA-mediated suppression of DSC2 expression increased cell motility. In E-cadherin-expressing ESCC cells, DSC2 restoration strengthened E-cadherin-mediated adherens junctions and promoted the localization of ß-catenin at these junctions, which indirectly inhibited ß-catenin-dependent transcription. These effects of DSC2 were not present in EC109 cells that lacked E-cadherin expression. ESCC patients with tumours that had reduced E-cadherin and negative DSC2 had poorer clinical outcomes than patients with tumours that lacked either E-cadherin or DSC2, implying that the invasive potential of ESCC cells was restricted by both DSC2 and E-cadherin-dependent junctions. Further studies revealed that DSC2 was a downstream target of miR-25. Enhanced miR-25 promoted ESCC cell invasiveness, whereas restoration of DSC2 abolished these effects. Collectively, our work suggests that miR-25-mediated down-regulation of DSC2 promotes ESCC cell aggressiveness through redistributing adherens junctions and activating beta-catenin signalling.

DACT2 is a candidate tumor suppressor and prognostic marker in esophageal squamous cell carcinoma.

In animals ranging from fish to mice, the function of DACT2 as a negative regulator of the TGF-ß/Nodal signal pathway is conserved in evolution, indicating that it might play an important role in human cancer. In this study, we showed that tumors with higher DACT2 protein level were correlated with better differentiation and better survival rate in patients with esophageal squamous cell carcinoma. Restored expression of DACT2 significantly inhibited growth, migration, and invasion of ESCC cells in vitro, and reduced tumorigenicity in vivo. Furthermore, when DACT2 expression was restored, the activity of TGF-ß/SMAD2/3 was suppressed via both proteasome and lysosomal degradation pathways, leading to F-actin rearrangement that might depend on the involvement of cofilin and ezrin-redixin-moesin (ERM) proteins. Taken together, we propose here that DACT2 serves as a prognostic marker that reduces tumor cell malignancy by suppressing TGF-ß signaling and promotes actin rearrangement in ESCC.

Involvement of Cyr61 in the growth, invasiveness and adhesion of esophageal squamous cell carcinoma cells.

Cysteine-rich 61 (Cyr61), a secreted protein which belongs to the CCN family, has been found to be differentially expressed in many cancers and to be involved in tumor progression. The expression of Cyr61 in esophageal squamous cell carcinoma (ESCC) has only recently been described, but the roles of Cyr61 in ESCC cells still remained unclear. In this study, we have shown that there are high levels of Cyr61 in ESCC cell lines. Furthermore, using RNA interference (RNAi), we stably silenced the expression of Cyr61 in EC109 cells, an ESCC cell line. The colony formation, MTT, cell migration, cell invasiveness and cell adhesion assays were employed to address the roles of Cyr61 in the growth, migration and adhesion of ESCC cells. The results have shown that Cyr61 knockdown by RNAi leads to a significant reduction of colony formation and cell growth. The migration and invasiveness ability of EC109 cells were also suppressed with the Cyr61 down-regulation. Furthermore, the adhesion of the EC109 cells was decreased in the Cyr61 knockdown cells compared to the control cells. Taken together, our data suggest that Cyr61 may play crucial roles in regulating neoplasm progression of ESCC.

Roles of ezrin in the growth and invasiveness of esophageal squamous carcinoma cells.

Ezrin, which crosslinks the cytoskeleton and plasma membrane, is involved in the growth and metastatic potential of cancer cells. Ezrin expression in esophageal squamous cell carcinoma (ESCC) was described recently, but its roles and the underlying mechanism(s) remain unclear. In our study, we first showed that ezrin in ESCC cell is expressed in the nucleus as well as in the cytoplasm and plasma membrane. Then, by using RNAi, we revealed that interference of ezrin expression suppressed the growth, adhesion and invasiveness of ESCC cells. Tumorigenesis experiments revealed that ezrin may directly regulate tumor formation in vivo. To explore the molecular mechanisms through which ezrin contributes to the proliferation and invasiveness of ESCC cells, we used cDNA microarrays to analyze ezrin knockdown cells and the control cells; of 39,000 genes examined, 297 were differentially expressed upon ezrin knockdown, including some proliferation- and invasiveness-related genes such as ATF3, CTGF and CYR61. Furthermore, pathway analysis showed that ezrin knockdown led to decreased activation of the TGF-beta and MAPK pathways, and ezrin-mediated cell invasiveness alteration was dependent on the activation of these pathways. Finally, immunohistochemical staining on 80 ESCC specimens and 50 normal esophageal mucosae revealed that the expression levels of 3 altered genes involved in the regulation of cell proliferation and tumor metastasis, including CTGF, CYR61 and ATF3, were altered in ESCCs, and their expression pattern correlated with ezrin expression. Taken together, we propose that ezrin might function in the growth and invasiveness of ESCC cells through the MAPK and TGF-beta pathways.