Sarcoma Cell Migration Research Articles

Combination of HDAC and FYN inhibitors in synovial sarcoma treatment.

The SS18-SSX fusion protein is an oncogenic driver in synovial sarcoma. At the molecular level, SS18-SSX functions as both an activator and a repressor to coordinate transcription of different genes responsible for tumorigenesis. Here, we identify the proto-oncogene FYN as a new SS18-SSX target gene and examine its relation to synovial sarcoma therapy. FYN is a tyrosine kinase that promotes cancer growth, metastasis and therapeutic resistance, but SS18-SSX appears to negatively regulate FYN expression in synovial sarcoma cells. Using both genetic and histone deacetylase inhibitor (HDACi)-based pharmacologic approaches, we show that suppression of SS18-SSX leads to FYN reactivation. In support of this notion, we find that blockade of FYN activity synergistically enhances HDACi action to reduce synovial sarcoma cell proliferation and migration. Our results support a role for FYN in attenuation of anti-cancer activity upon inhibition of SS18-SSX function and demonstrate the feasibility of targeting FYN to improve the effectiveness of HDACi treatment against synovial sarcoma.

Identifying specific TLS-associated genes as potential biomarkers for predicting prognosis and evaluating the efficacy of immunotherapy in soft tissue sarcoma.

The tertiary lymphatic structure (TLS) is an important component of the tumor immune microenvironment and has important significance in patient prognosis and response to immune therapy. However, the underlying mechanism of TLS in soft tissue sarcoma remains unclear. A total of 256 RNAseq and 7 single-cell sequencing samples were collected from TCGA-SARC and GSE212527 cohorts. Based on published TLS-related gene sets, four TLS scores were established by GSVA algorithm. The immune cell infiltration was calculated via TIMER2.0 and "MCPcounter" algorithms. In addition, the univariate, LASSO, and multivariate-Cox analyses were used to select TLS-related and prognosis-significant hub genes. Single-cell sequencing dataset, clinical immunohistochemical, and cell experiments were utilized to validate the hub genes. In this study, four TLS-related scores were identified, and the total-gene TLS score more accurately reflected the infiltration level of TLS in STS. We further established two hub genes (DUSP9 and TNFSF14) prognosis markers and risk scores associated with soft tissue sarcoma prognosis and immune therapy response. Flow cytometry analysis showed that the amount of CD3, CD8, CD19, and CD11c positive immune cell infiltration in the tumor tissue dedifferentiated liposarcoma patients was significantly higher than that of liposarcoma patients. Cytological experiments showed that soft tissue sarcoma cell lines overexpressing TNFSF14 could inhibit the proliferation and migration of sarcoma cells. This study systematically explored the TLS and related genes from the perspectives of bioinformatics, clinical features and cytology experiments. The total-gene TLS score, risk score and TNFSF14 hub gene may be useful biomarkers for predicting the prognosis and immunotherapy efficacy of soft tissue sarcoma.

The expression of neuraminidase-1 in Ewing sarcoma and its impact on sarcoma cell proliferation and migration

Objective: To investigate the expression of neuraminidase-1 (NEU1) in Ewing sarcoma (ES) tissue and its effect on the proliferation and migration of ES cells. Methods: To obtain datasets of ES from the National Center for Biotechnology Information's High-Throughput Gene Expression Omnibus (GEO) for the analysis of NEU1 expression in ES; to acquire ES patient dataset from the International Cancer Genome Consortium (ICGC) database and apply Kaplan-Meier survival analysis to investigate the relationship between NEU1 and the prognosis of ES patients; adopting both univariate and multivariate Cox regression analysis to determine whether NEU1 is a prognostic factor for ES; adopting the Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation to analyze the potential mechanism of NEU1 in regulating the malignant biological behavior of ES; adopting the real-time fluorescence quantitative polynucleotide chain reaction (RT-qPCR) to verify the expression of NEU1 in the human bone marrow mesenchymal stem cells (hBMSC) and the ES cell line RD-ES; adopting the transfection technology to knock down the expression of NEU1 in ES cell lines and divide them into two groups: shRNA-NEU1 and shRNA-NC to explore the effects of altered NEU1 expression on ES malignant behavior; adopting the cell counting kit (CCK-8) and cell clone formation experiment to detect the proliferation ability of two groups of cells; adopting the scratch healing experiment to test the cell migration ability of the two groups. Results: We retrieved and analyzed data from the GEO database, including GSE17674 (44 ES tissues and 18 normal tissues) and GSE17679 (87 ES tissues and 18 normal tissues), and found that NEU1 expression was significantly higher in ES tissues compared to normal control tissues (P<0.001). The complete gene expression and clinical information of 56 ES patients obtained from the ICGC database revealed that the ES patients with high NEU1 expression (n=28) had a significantly lower overall survival rates at different time points compared to those with low NEU1 expression (n=28) (HR=2.830, 95%CI:1.324-6.051, P=0.005). Univariate analysis indicated that NEU1 could impact ES patient prognosis (HR=1.049, 95%CI: 1.008-1.092, P=0.019), and multivariate analysis further suggested that NEU1 could serve as a risk factor for ES prognosis (HR=1.087, 95%CI: 1.028-1.148, P=0.003). KEGG results show that MAPK signaling pathway and cell adhesion molecule signaling pathway were potential mechanisms regulating the malignant process of ES. The RT-qPCR results showed that the expression level of NEU1 in the RD-ES cell line is significantly higher than that in the control cell hBMSC (2 184.23±527.32 vs 1.00±0.08, P<0.001). The CCK-8 experiment results show that the proliferation of RD-ES cells in the NEU1 knockdown group was lower than that in the control group at 24, 48, and 72 hours (0.494±0.126 vs 0.696±0.118, 0.657±0.096 vs 1.142±0.182, 1.053±0.064 vs 1.980±0.146, all P<0.001). The results of single cell clone formation experiment show that the number of colony formation in the low expression NEU1 group was significantly lower than that in the control group (184.2±123.9 vs 362.8±78.0, P=0.021). The cell scratch healing experiment finds that the average scratch distance of the NEU1 knockdown group was significantly lower than that of the control group (19.6%±5.7% vs 56.0%±7.6%, P<0.001). Conclusion: NEU1 may be a prognostic factor in ES, and its abnormal expression in ES can affect the proliferation and migration ability of the ES cells, leading to poor prognosis in ES patients.

Extracellular vesicle-associated IGF2BP3 tunes Ewing sarcoma cell migration and affects PI3K/Akt pathway in neighboring cells

Ewing sarcoma (EWS) is a challenging pediatric cancer characterized by vast intra-tumor heterogeneity. We evaluated the RNA-binding protein IGF2BP3, whose high expression correlates with a poor prognosis and an elevated tendency of metastases, as a possible soluble mediator of inter-cellular communication in EWS. Our data demonstrate that (i) IGF2BP3 is detected in cell supernatants, and it is released inside extracellular vesicles (EVs); (ii) EVs from IGF2BP3-positive or IGF2BP3-negative EWS cells reciprocally affect cell migration but not the proliferation of EWS recipient cells; (iii) EVs derived from IGF2BP3-silenced cells have a distinct miRNA cargo profile and inhibit the PI3K/Akt pathway in recipient cells; (iv) the 11 common differentially expressed miRNAs associated with IGF2BP3-positive and IGF2BP3-negative EVs correctly group IGF2BP3-positive and IGF2BP3-negative clinical tissue specimens. Overall, our data suggest that IGF2BP3 can participate in the modulation of phenotypic heterogeneity.

The Roles of Exosomes in Metastasis of Sarcoma: From Biomarkers to Therapeutic Targets

Sarcoma is a heterogeneous group of mesenchymal neoplasms with a high rate of lung metastasis. The cellular mechanisms responsible for sarcoma metastasis remain poorly understood. Furthermore, there are limited efficacious therapeutic strategies for treating metastatic sarcoma. Improved diagnostic and therapeutic modalities are of increasing importance for the treatment of sarcoma due to their high mortality in the advanced stages of the disease. Recent evidence demonstrates that the exosome, a type of extracellular vesicle released by virtually all cells in the body, is an important facilitator of intercellular communication between the cells and the surrounding environment. The exosome is gaining significant attention among the medical research community, but there is little knowledge about how the exosome affects sarcoma metastasis. In this review, we summarize the multifaceted roles of sarcoma-derived exosomes in promoting the process of metastasis via the formation of pre-metastatic niche (PMN), the regulation of immunity, angiogenesis, vascular permeability, and the migration of sarcoma cells. We also highlight the potential of exosomes as innovative diagnostic and prognostic biomarkers as well as therapeutic targets in sarcoma metastasis.

CNTNAP4 signaling regulates osteosarcoma disease progression

Improved treatment strategies for sarcoma rely on clarification of the molecular mediators of disease progression. Recently, we reported that the secreted glycoprotein NELL-1 modulates osteosarcoma (OS) disease progression in part via altering the sarcomatous extracellular matrix (ECM) and cell-ECM interactions. Of known NELL-1 interactor proteins, Contactin-associated protein-like 4 (Cntnap4) encodes a member of the neurexin superfamily of transmembrane molecules best known for its presynaptic functions in the central nervous system. Here, CRISPR/Cas9 gene deletion of CNTNAP4 reduced OS tumor growth, sarcoma-associated angiogenesis, and pulmonary metastases. CNTNAP4 knockout (KO) in OS tumor cells largely phenocopied the effects of NELL-1 KO, including reductions in sarcoma cell attachment, migration, and invasion. Further, CNTNAP4 KO cells were found to be unresponsive to the effects of NELL-1 treatment. Transcriptomic analysis combined with protein phospho-array demonstrated notable reductions in the MAPK/ERK signaling cascade with CNTNAP4 deletion, and the ERK1/2 agonist isoproterenol restored cell functions among CNTNAP4 KO tumor cells. Finally, human primary cells and tissues in combination with sequencing datasets confirmed the significance of CNTNAP4 signaling in human sarcomas. In summary, our findings demonstrate the biological importance of NELL-1/CNTNAP4 signaling axis in disease progression of human sarcomas and suggest that targeting the NELL-1/CNTNAP4 signaling pathway represents a strategy with potential therapeutic benefit in sarcoma patients.

Autophagy inhibitor potentiates the antitumor efficacy of apatinib in uterine sarcoma by stimulating PI3K/Akt/mTOR pathway.

The present study aims to examine the effects of apatinib combined with autophagy inhibitor 3-Methyladenine (3-MA) on the proliferation and apoptosis of human uterine sarcoma in FU-MMT-1 and MES-SA cells and its tumor inhibition effect in xenograft model of uterine sarcoma. Different concentrations of 3-MA and apatinib were used to treat the uterine sarcoma cell lines (MES-SA and FU-MMT-1 cells). The cell viability was detected by CCK8 method. Flow cytometry was used to detect the apoptosis and cell cycle. Wound closure assay and Transwell assay were performed to measure the migration ability of cells. Western blot was used to determine the apoptosis proteins and autophagy proteins. A nude mice sarcoma xenograft model was established and treated with apatinib alone, 3-MA alone, or combined incubation of them. Tumor size of xenograft and the mice survival rate were measured. Combination of 3-MA and apatinib significantly inhibited the proliferation and migration ability, but increased the apoptosis rate of uterine sarcoma cells compared to apatinib. The combination of 3-MA and apatinib significantly limited the tumor size of xenograft and increased the survival rate of mice compared to apatinib alone. Apatinib inhibited the PI3K/Akt/mTOR pathway, while 3-MA and the combination of 3-MA and apatinib significantly activated the PI3K/Akt/mTOR pathway and inhibited autophagy. Combination of 3-MA and apatinib increased apoptosis compared to apatinib alone. The expression of VEGFR-2 was not impacted by 3-MA. Combination of apatinib and autophagy inhibitor 3-MA significantly inhibited the growth and migration of uterine sarcoma cells and xenograft. Autophagy inhibition may increase the antitumor effect of apatinib via the PI3K/Akt/mTOR pathway.

LncRNA TUG1 promotes Ewing's sarcoma cell proliferation, migration, and invasion via the miR-199a-3p-MSI2 signaling pathway.

The aim of this study was to investigate the roles and potential mechanisms of long non-coding RNA (lncRNA) taurine upregulated gene 1 (TUG1) in the proliferation, migration, and invasion of Ewing's sarcoma cells. RT-qPCR was used to detect the expression of TUG1, microRNA-199a-3p (miR-199a-3p), and musashi2 (MSI2) in Ewing's sarcoma tissues and cell lines. Kaplan-Meier overall survival curves showed the survival rates of Ewing's sarcoma patients with high and low expression of TUG1. The association between the expressions of TUG1/MSI2 and miR-199a-3p in Ewing's sarcoma tissues was assessed by Pearson's correlation analysis. Cell proliferation, migration, and invasion were detected by CCK-8 assay and Transwell assay, respectively. The protein level of MSI2 was determined using western blotting. The interaction between TUG1/MSI2 and miR-199a-3p was validated by the dual-luciferase reporter assay. The levels of TUG1 and MSI2 were increased, while the level of miR-199a-3p was decreased in Ewing's sarcoma tissues and cells. High expression of TUG1 or MSI2 indicated a decreased overall survival rate of Ewing's sarcoma patients. TUG1/MSI2 level was negatively correlated with miR-199a-3p level. While TUG1 level was positively correlated with MSI2 level. In Ewing's sarcoma cells, knockdown of TUG1/MSI2 or overexpression of miR-199a-3p inhibited cell proliferation, migration, and invasion, whereas the overexpression of TUG1/MSI2 presented the opposite results. TUG1 functioned as a competing endogenous RNA to regulate MSI2 expression by sponging miR-199a-3p. Finally, miR-199a-3p inhibitor or MSI2 overexpression counteracted the TUG1 knockdown-mediated inhibitory effect on Ewing's sarcoma cell proliferation, migration, and invasion. TUG1 promotes proliferation, migration, and invasion of Ewing's sarcoma cells via sequestering miR-199a-3p to enhance the MSI2 expression, suggesting that TUG1 might be a potential target for treating Ewing's sarcoma.

Rab11-mediated focal adhesion turnover in sarcoma cell migration.

Focal adhesion (FA) turnover has been demonstrated to play an important role in cell migration; however, the mechanism of FA turnover is complicated and requires further investigation. In this study, Rab11, which is involved in endosome recycling, was examined in terms of a direct regulatory function in FA formation during cell migration. Wild-type and dominant negative (DN) Rab11 or shRab11 were transfected into human HT1080 fibrosarcoma cells; the cell motility and migration abilities were determined, and localization of Rab11 and FA molecules was monitored by confocal microscopy. The results showed that Rab11 deficiency or the DN form inhibited sarcoma cell migration. Rab11 was also found to be co-localized with recycled β1 integrin and affected FA formation. We further employed immunofluorescence and immunoprecipitation to examine the physical interaction between Rab11 and focal adhesion kinase (FAK), and the results suggested that Rab11 affected cell migration by regulating FAK recycling to aid formation of an FA complex on the cell membrane.

Abstract 6594: Targeting Ewing sarcoma and osteosarcoma with anti-MCAM chimeric antigen receptor modified NK cells

Abstract Ewing sarcoma and osteosarcoma are the two most common pediatric malignant bone tumors. Patients who present with metastatic disease or relapse have a dismal average 5-year survival of only 20%. Novel therapeutic approaches are desperately needed. The melanoma cell adhesion molecule (MCAM/CD146/MUC18) is highly expressed in sarcoma and metastatic pediatric tumors. MCAM depletion inhibits Ewing sarcoma cell migration in vitro and tumor metastasis in vivo. High MCAM levels in patient tumors are associated with poor survival in Ewing sarcoma. MCAM therefore constitutes a unique and novel target for immunotherapy against MCAM+ sarcomas. We previously demonstrated that natural killer (NK) cell engineered to express chimeric antigen receptor (CAR) against CD20 significantly reduced tumor burden, prevented tumor dissemination, and extended survival of treated mice compared to mock NK treatment in humanized Burkitt lymphoma xenograft mouse model. We hypothesize that anti-MCAM CAR NK cell will enhance the inherent NK-sarcoma cytotoxicity by better targeting sarcoma cells. Here we develop an anti-MCAM CAR NK cell and investigate its efficacy in promoting NK cell cytotoxicity against Ewing sarcoma and osteosarcoma. MCAM expression in sarcoma cell lines and tumor samples were validated by flow cytometry and western blotting. Peripheral blood mononuclear cells (PBMCs) are expanded into NK cells ex vivo using K562-mbIL21-41BBL artificial antigen presenting cells (aAPC). Anti-MCAM CAR NK cells are generated by non-viral electroporation of anti-MCAM CAR mRNA into expanded NK cells. Anti-MCAM CAR NK cytotoxicity is evaluated in vitro by luciferase based cytotoxicity assay. We detected various levels of MCAM expression in Ewing sarcoma and osteosarcoma cell lines and tumors. In addition, we noticed an interesting patchy expression pattern of MCAM in the Ewing sarcoma tumor sample. Importantly, we found a significantly increased cytotoxicity of anti-MCAM CAR NK cells compared to mock NK cells (59.9±10 vs 13.3±3.7 at E:T ratio of 5:1, p=0.006; and 65.6±7 vs 13.7±3.4 at 10:1, p=0.004) against Ewing sarcoma TC71 cells after co-culturing for 4 hours. A similar result was observed at the time point of 48 hours (91.5±8 vs 59±0.5 at E:T of 5:1, p=0.018). Anti-MCAM CAR NK cells also showed significantly enhanced cytotoxicity against U2OS osteosarcoma cells compared to mock NK cells (32.6±23.2 vs 0.5±19.5 at E:T of 0.5:1, p=0.004; 47.1±27.4 vs 10.2±24 at E:T of 1:1, p=0.001) at the time point of 48 hours. In summary, these findings demonstrated enhanced efficacy of anti-MCAM CAR NK cells compared to mock NK cells against Ewing sarcoma and osteosarcoma cells and support a further preclinical investigation whether targeting MCAM by CAR NK will limit sarcoma growth and/or metastasis and increase survival in vivo. Citation Format: Aliza Gardenswartz, Wen Luo, Jeremy M. Rosenblum, Janet Ayello, Mitchell S. Cairo. Targeting Ewing sarcoma and osteosarcoma with anti-MCAM chimeric antigen receptor modified NK cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6594.

Differential gene expression identifies KRT7 and MUC1 as potential metastasis-specific targets in sarcoma.

BackgroundDespite numerous discoveries regarding the molecular genesis and progression of primary cancers, the biology of metastasis remains poorly understood. Compared to very large numbers of circulating tumor cells that are now known to accompany nearly all cancers, a relatively limited number of lesions actually develop in most patients with metastases. We hypothesized that phenotypic changes driven by differential gene expression in a finite subpopulation of tumor cells render those cells capable of metastasis and sought to identify key pathways through analysis of gene expression in primary and metastatic lesions from the same patients.MethodsWe compared whole-genome expression in 4 matched samples of primary and metastatic sarcoma, then evaluated candidate genes with differential expression via quantitative PCR in 30 additional matched sets, tumor tissue immunostaining, siRNA loss-of-function in a sarcoma cell migration assay, and clinical correlation with overall and disease-free survival after metastasectomy.ResultsComparison of microarray signals identified differential expression of cell adhesion genes, including upregulation of KRT7 and MUC1 in metastases; KRT7 and MUC1 upregulation was confirmed in 22 (73%) and 20 (67%) matched sets of metastatic/primary tumors, respectively. Silencing of KRT7 and MUC1 via targeted siRNAs suppressed sarcoma cell migration in vitro, and a significant correlation (two-sided) was observed between both KRT7 and MUC1 expression in metastases and overall patient survival.ConclusionKRT7 and MUC1 may play a significant role in enabling sarcoma metastasis, and they may therefore be important prognostic biomarkers as well as potential targets for therapeutic prevention of metastasis.

MiR-9 promotes synovial sarcoma cell migration and invasion by directly targeting CDH1.

Invasion and metastasis of synovial sarcoma is the leading cause of death in patients. Epithelial mesenchymal transition (EMT) accelerates tumor cell invasion and metastasis. MiR-9 promotes tumor metastasis by inducing EMT. However, the role of miR-9 in synovial sarcoma is still not clear. Overexpression or knockdown of miR-9 in human synovial sarcoma (HSS) cell lines was carried out by miR-9 mimics or miR-9 inhibitors transfection. Cell proliferation, apoptosis, migration and invasion were detected using MTS and colony formation assays, flow cytometry, wound healing and transwell assays, respectively. Luciferase reporter assay was applied to study the interaction between miR-9 and CDH1. Nude mice xenograft model was established, and immunohistochemistry staining assessed Ki-67 level. The related mRNA and protein expression levels were evaluated by qRT-PCR and Western blotting. The bioinformatics analyses and luciferase reporter assay showed that miR-9 can target CDH1 3'-UTR. Moreover, miR-9 could induce EMT of HSS cells via targeting CDH1. The negative regulation of miR-9 on CDH1 expression was also confirmed in a mouse xenograft model of synovial sarcoma. Furthermore, miR-9 was observed to induce HSS cell proliferation, migration and invasion and inhibit apoptosis. MAPK/ERK and Wnt/β-catenin signal pathways were activated by the miR-9 overexpression in HSS cells, and then further enhancing tumorigenesis of HSS, which was further confirmed in the mouse model. MiR-9 induces EMT by targeting CDH1, and activates MAPK/ERK and Wnt/β-catenin signal pathways, thus promoting HSS tumorigenesis.

Abstract A02: Targeting EWS-FLI1 dose-dependent epithelial-to-mesenchymal transition

Abstract Reversible transitions from a proliferative to a nonproliferative but highly migratory and invasive phenotype, widely known as epithelial-to-mesenchymal transition (EMT), is a prerequisite for wound healing and organ development but also a hallmark of cancer. Recently, phenotypic changes resembling EMT were described to occur in Ewing sarcoma upon modulation of expression of the driver oncogene EWS-FLI1. Evidence was provided for the presence of highly migratory EWS-FLI1-low cells in Ewing sarcoma cell lines as well as in about 20% of primary tumors at low frequency (Franzetti et al., 2017). Importantly, when injected into the tail vein of immunosuppressed mice, EWS-FLI1-low cells were more likely to result in lung metastases than EWS-FLI1-high cells upon re-expression of the oncogene. In our study, we identified a mechanism for the cytoskeletal reprograming underlying EWS-FLI1 dose-dependent phenotypic transitions in Ewing sarcoma. We provide evidence for EWS-FLI1 blocking access of the transcriptional coactivators MRTFB and TAZ to the YAP/TEAD transcription factor complex on enhancers of EWS-FLI1 anti-correlated genes. Upon modulation of EWS-FLI1 expression, TEADs get activated downstream of Rho signaling through recruitment of MRTFB/YAP/TAZ, leading to the expression of key cytoskeletal components and regulators involved in the reorganization of the actin cytoskeleton. Based on these findings, we hypothesized that small-molecule inhibitors of the YAP/TAZ/TEAD pathway should interfere with the migratory phenotype of EWS-FLI1-low cells. In fact, the YAP/TAZ inhibitor verteporfin (Visudyne®), which is clinically used as a photosensitive drug in the treatment of age-related macular degeneration, was able to block Ewing sarcoma cell migration at low nanomolar concentrations without affecting tumor cell proliferation. These results suggest that YAP/TAZ inhibitors may be considered as a promising add-on to conventional combination treatments to prohibit tumor cell dissemination and metastasis. Citation Format: Anna M. Katschnig, Maximilian O. Kauer, Raphaela Schwentner, Eleni M. Tomazou, Dave N.T. Aryee, Heinrich Kovar. Targeting EWS-FLI1 dose-dependent epithelial-to-mesenchymal transition [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A02.

MicroRNA‑34b promotes proliferation, migration and invasion of Ewing's sarcoma cells by downregulating Notch1.

Ewing's sarcoma is the second most frequent bone and soft tissue sarcoma, which is commonly driven by the Ewing's sarcoma breakpoint region 1-friend leukemia integration 1 transcription factor (EWS-FLI1) fusion gene. Since microRNAs (miRs) can act as either oncogenes or tumor suppressor genes in human cancer, and miR-34b has been reported to act as a tumor suppressor, the role of miR-34b in Ewing's sarcoma was investigated in the present study. The results demonstrated that miR-34b expression levels were higher in tumor samples compared within normal tissue samples. Notably, miR-34b expression levels were significantly higher in EWS-FLI1-positive samples compared within EWS-FLI1-negative samples. The effects of miR-34b expression on cell proliferation, migration and invasion were also examined. miR-34b expression was inhibited using small interfering (si)RNA targeting the fusion gene. Transfection of a miR-34b precursor sequence into siRNA-treated tumor cells resulted in a significant increase in cell growth, migration and invasion compared within the control group. In addition, the adhesive ability was increased in the Ewing's sarcoma cell line RD-ES, but not A673, following miR-34b upregulation. Conversely, downregulation of miR-34b expression led to a significant decrease in cell growth, migration and invasion. Notch has previously been reported to serve either oncogenic or tumor suppressive roles in human cancer. The results indicated that Notch1 and its target genes, Hes family BHLH transcription factor 1 and Hes-related family BHLH transcription factor with YRPW motif 1, were suppressed by miR-34b directly In conclusion, EWS-FLI1 may modulate miR-34b expression directly or indirectly, and miR-34b potentially has an oncogenic role in Ewing's sarcoma by downregulating Notch1.

Abstract 5163: Rab11 regulates focal adhesion kinase dynamics in sarcoma cell migration

Abstract Focal adhesion (FA) turnover has been demonstrated to play an important role in controlling cell migration and cancer metastasis. FA disassembly was shown to be regulated by endocytosis during cell migration. Rabs are a kind of GTPase that participate in various cellular vesicle transports; however, whether the Rabs play a role in mediating vesicle transport for FA formation is not clear. This study focuses on Rab11, which plays a role in endosome recycling, and examines whether Rab11 has regulatory function in FA formation during cell migration. Wild-type and deficient Rab11 were transfected into human HT1080 fibrosarcoma cells, the cell migration ability was determined by trans-well assay, the localization of Rab11 and focal adhesion molecules were monitored by confocal microscopy and the in vivo study was carried out with subcutaneous xenograft mouse model to evaluate the effect of Rab11 on tumor growth. Results showed Rab11 deficiency inhibited sarcoma cell migration; the Rab11 was also found colocalized with recycled β1 integrin and affected focal adhesion formation. The live image results demonstrated the inactive GDP form Rab11 has no effect on random cell migration; however, the Rab11 knockdown inhibited random cell migration. We further used immunofluorescence and immunoprecipitation and identified the physical interaction of Rab11 and FAK. In the in vivo study, wild-type Rab11 transfected cells increased the tumor volume in xenograft mouse, but not in Rab11-deficient cells. Taken together, the results suggested Rab11 affected cell migration by regulating focal adhesion dynamics through integrin recycling and the effect of Rab11 may be through protein-protein interaction other than GTP activation. Citation Format: Ling-Yi Kao, Wan-Chen Wei, Hsiang-Ling Chiu, Yi-Che Wu, Wei-Ting Chao. Rab11 regulates focal adhesion kinase dynamics in sarcoma cell migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5163.

Abstract 2315: Inhibition of cyclin-dependent kinase 4 as a potential therapeutic strategy for treatment of synovial sarcoma

Abstract Synovial sarcoma is a rare and aggressive form of soft tissue cancer that affects the extremities of the arms or legs, for which current chemotherapeutic agents have not been proven to be very effective. The cyclin-dependent kinase 4/6-retinoblastoma protein (CDK4/6-Rb) pathway is aberrant in a large proportion of cancer. Recent evidences on pre-clinical application of CDK4 inhibitors have been implicated in many types of human cancers, and the FDA has approved the CDK4 selective inhibitor for the treatment of breast cancer. However, the expression and therapeutic potential of CDK4 in synovial sarcoma remain unclear. In the present study, we examined the expression of CDK4 in synovial sarcoma cell lines by western blot and immunofluorescence assay, and in synovial sarcoma tissue microassays by immunohistochemical analysis. Cell viabilities were determined by MTT assay after exposure to different dosages of the selective CDK4 inhibitor. Flow cytometry analysis and wound healing assay were conducted to determine the mechanisms underlying the cytotoxic effects of the selective CDK4 inhibitor. CDK4 specific small interference RNA was used to validate the effect of targeting CDK4 by the selective CDK4 inhibitor in synovial sarcoma cells. We found that CDK4 was highly expressed in human synovial sarcoma, and was related to clinical stage and TNM grade in synovial sarcoma patients and poor prognosis in sarcoma patients. Cell viabilities determined by MTT assay after exposure to different dosages of the selective CDK4 inhibitor showed that this selective CDK4 inhibitor repressed synovial sarcoma cell proliferation and growth in a dose- and time- dependent manner. The selective CDK4 inhibitor inhibited the CDK4/6-Rb signaling pathway and promoted cell apoptosis without influence on the expression of CDK4/6, suggesting that the selective CDK4 inhibitor only repressed the hyperactivation, not the production of CDK4/6. The inhibition effect of the selective CDK4 inhibitor was confirmed by knockdown of CDK4 with specific small interference RNA. Flow cytometry analysis revealed that the selective CDK4 inhibitor induced G1 cell-cycle arrest by targeting CDK4/6-Rb pathway in synovial sarcoma cells. Furthermore, the wound healing assay exhibited that inhibition of CDK4/6-Rb pathway with the use of the selective CDK4 inhibitor significantly decreased synovial sarcoma cell migration in vitro. Our data highlight the role of dysregulated CDK4/6-Rb pathway and current selective CDK4/6 inhibitor may be a potential promising therapeutic agent in the targeted treatment of human synovial sarcoma. Citation Format: Xiaoyang Li, Cassandra Garbutt, Francis John Hornicek, Zhenfeng Duan. Inhibition of cyclin-dependent kinase 4 as a potential therapeutic strategy for treatment of synovial sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2315.

Inhibition of cyclin-dependent kinase 4 as a potential therapeutic strategy for treatment of synovial sarcoma

Synovial sarcoma is a highly aggressive but rare form of soft tissue malignancy that primarily affects the extremities of the arms or legs, for which current chemotherapeutic agents have not been proven to be very effective. The cyclin-dependent kinase 4/6-retinoblastoma protein (CDK4/6-Rb) pathway of cell cycle control is known to be aberrant in a large proportion of cancers. Recently, CDK4 inhibitors have successfully been used pre-clinically for the treatment of many human cancers, and in 2015, following the success of clinical trials, the FDA approved the first selective CDK4/6 inhibitor, palbociclib, for the treatment of endocrine therapy resistant breast cancers. However, the expression and therapeutic potential of targeting CDK4 in synovial sarcoma remains unclear. In the present study, we report that CDK4 is highly expressed in human synovial sarcoma, and high CDK4 expressions are associated with poor prognosis in sarcomas patients and the clinical stage and the TNM grade in synovial sarcoma patients. Knockdown of CDK4 with specific small interference RNAs inhibits cell proliferation and enhances apoptotic effects in synovial sarcoma cells. CDK4 inhibitor palbociclib suppresses synovial sarcoma cell proliferation and growth in a dose and time-dependent manner. Palbociclib also inhibits the CDK4/6-Rb signaling pathway and promotes cell apoptosis without changing CDK4/6 protein levels, suggesting that palbociclib only represses the hyper-activation, not the expression of CDK4/6. Flow cytometry analysis reveals that palbociclib induces G1 cell-cycle arrest and apoptotic effects by targeting the CDK4/6-Rb pathway in synovial sarcoma cells. Furthermore, wound healing assays demonstrate that inhibition of the CDK4/6-Rb pathway by palbociclib significantly decreases synovial sarcoma cell migration in vitro. Our study highlights the importance of the CDK4/6-Rb pathway in human synovial sarcoma pathogenesis, and the role of the current selective CDK4/6 inhibitor, palbociclib, as a potential promising targeted therapeutic agent in the treatment of human synovial sarcoma.

Effects of baicalein on proliferation, apoptosis, migration and invasion of Ewing's sarcoma cells.

Ewing's sarcoma (ES) is a rare tumor that is more frequent in pediatric and adolescent age groups. In the past few decades, long-term survival in affected patients has improved due to the success of multimodal therapy. However, long-term survival is inevitably restricted by the late side-effects of chemotherapy. Besides, early metastasis also contributes to the poor prognosis of ES. Recently, traditional Chinese medicines (TCMs) have increasingly attracted interest due to the promising clinical results and fewer side-effects for the treatment of cancers. Among the various TCMs, the root of Scutellaria baicalensis exerts anti-inflammatory properties as a well-known herb in traditional Chinese medicine. Baicalein (5,6,7-trihydroxyflavone) derived from the root of Scutellaria baicalensis is a bioactive compound, which possesses a powerful pro-apoptotic activity in various cancers such as hepatocellular carcinoma and myeloma. However, the effects of baicalein on ES cells remain still unknown. We anticipated that baicalein also has apoptotic activity in ES. The aim of the present study was to investigate the effects of baicalein on viability, apoptosis, migration and invasion of ES cells, and further to elaborate the molecular mechanism of baicalein-induced ES cell apoptosis. We found that baicalein markedly inhibited ES cells viability in a time- and dose-dependent manner, especially SK-ES-1 cells and could promote the apoptosis of ES cells. Additionally, baicalein was capable of upregulating the expression of the pro-apoptotic proteins Bax and cytochromec, reducing the expression of the anti-apoptotic protein Bcl-2, elevating the ratio of Bax/Bcl-2, and triggering the mitochondrial apoptotic pathway, which led to caspase-3 and caspase-9 activation and PARP cleavage. Meanwhile, the activation of caspase-8 and the death receptor pathway was also observed. Besides, baicalein could reduce ES migration and invasion invitro, which showed its potential to inhibit ES metastasis, besides contributing to the decrease in the expression of matrix metalloproteinases (MMP)-2 and MMP-9. In conclusion, baicalein has a potent tumor-suppressor activity by inducing cell apoptosis through the mitochondrial apoptotic pathway and the death receptor pathway in ES cells, thus it may serve as a novel and effective candidate agent for ES treatment.

Abstract 5477: Hydrogels to study ECM-oxygen gradient interactions for sarcoma cell migration

Abstract Hypoxia is a critical factor in the progression and metastasis of many cancers, including soft tissue sarcomas. Frequently, oxygen (O2) gradients develop in tumors as they grow beyond their vascular supply, leading to heterogeneous areas of O2 depletion. Here, we report the impact of hypoxic O2 gradients and collagen fibers on sarcoma cell invasion and migration. We begun with O2 gradient measurements and found that large sarcoma mouse tumors (&amp;gt;300 mm3) contain a severely hypoxic core [≤0.1% partial pressure of O2 (pO2)] whereas smaller tumors possessed hypoxic gradients throughout the tumor mass (0.1–6% pO2). To analyze tumor invasion, we used O2-controllable hydrogels to recreate the physiopathological O2 levels in vitro. Small tumor grafts encapsulated in the hydrogels revealed increased invasion that was both faster and extended over a longer distance in the hypoxic hydrogels compared with nonhypoxic hydrogels. To model the effect of the O2 gradient accurately, we examined individual sarcoma cells embedded in the O2-controllable hydrogel. We observed that hypoxic gradients guide sarcoma cell motility and matrix remodeling through hypoxia-inducible factor-1α (HIF-1α) activation. We further found that in the hypoxic gradient, individual cells migrate more quickly, across longer distances, and in the direction of increasing O2 tension. Treatment with minoxidil, an inhibitor of hypoxia-induced sarcoma metastasis, abrogated cell migration and matrix remodeling in the hypoxic gradient. Next, we have developed hypoxic hydrogels where we can control collagen fiber density and cross-linking density independently from oxygen gradients. Using this new platform we show that an increase in fiber density in conjunction with the hypoxic oxygen gradient leads to an increase in sarcoma cell migration. This increase in migration speed was also correlated with quicker stress relaxation times of the collagen gel and larger pore size. Using these platforms we are able to create a novel 3D experimental system with control over mechanical properties (crosslinking density, binding sight density, fiber density, stress relaxation time, and stiffness) independently from oxygen concentration. Overall, we show that O2 acts as a 3D physicotactic agent during sarcoma tumor invasion and that this migration is modulated by collagen fiber density and pore size, in the surrounding matrix. We propose the O2-controllable hydrogels as a predictive system to study early stages of the metastatic process and therapeutic targets. Citation Format: Daniel M. Lewis, Sharon Gerecht, T. S. Karin Eisinger-Mathason. Hydrogels to study ECM-oxygen gradient interactions for sarcoma cell migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5477. doi:10.1158/1538-7445.AM2017-5477

Collagen Fiber Architecture Regulates Hypoxic Sarcoma Cell Migration.

Collagen is prevalent in the microenvironment of many cancer types and has been demonstrated to play an important role during disease progression. We previously showed the importance of hypoxic gradients in sarcoma cell migration. Here, we utilized an oxygen gradient collagen gel platform to determine the impact of collagen fiber density and hypoxic gradient on sarcoma cell migration. The oxygen gradient was created by regulating the oxygen diffusion coefficient along with the cellular oxygen consumption rate. Collagen fiber density in the hydrogels is modified by changing the preincubation period of the collagen gel solution at 4 °C, controlling fiber density independently of collagen concentration and oxygen tension. High fiber density gels have wider and longer fibers but a similar microscale pore size with a larger nanoscale pore size and quicker stress relaxation time, compared to the low fiber density gel. Both gels have the same Young's modulus. We analyzed responses of sarcoma cells encapsulated in the different hydrogels for 3 days. In the nonhypoxic low fiber density constructs, sarcoma cells exhibit a larger aspect ratio, and the matrix has less fiber alignment compared to the nonhypoxic high fiber density constructs. Interestingly, we found a minimal effect of fiber density on cell migration and the ability of the cells to degrade the matrix in nonhypoxic constructs. When compared with hypoxic constructs, we observed the opposite trend, where cells in low fiber density constructs exhibit a lower aspect ratio and the matrix has more aligned fibers compared to hypoxic high fiber density constructs. Sarcoma cells encapsulated in high fiber density hypoxic gels migrated faster and degraded the matrix more rapidly compared to the low fiber density hypoxic constructs. Overall, we show that hypoxic cell migration and matrix degradation are enhanced in high fiber density gels, while hypoxic matrix alignment is prominent in low fiber density gels. Our results suggest that the differences in cellular responses under hypoxic gradients are due to the hydrogel architecture including fiber density, size (length and width), and stress relaxation.