Article Figures & Data
Figures
- Figure 1.
E-cadherin upregulation is prognostic for improved outcomes in sarcoma. A and B, Osteosarcomas with elevated E-cadherin have better metastasis-free survival (A) and overall survival (B) as compared to tumors with low/no E-cadherin expression. C and D, Soft-tissue sarcomas (STS) from The Cancer Genome Atlas with higher E-cadherin mRNA (C) and protein expression (D) have improved overall survival as compared to tumors with low or no E-cadherin.
- Figure 2.
Ectopic E-cadherin expression in sarcoma cells does not alter EMT. A, Ectopic expression of E-cadherin in 143B human osteosarcoma cells has no influence on mesenchymal markers (Snail, Slug, Twist, Zeb1, Vimentin). B, E-cadherin expression has no effect on migration of 143B cells. C, Images in B were collected every 2 hours and quantified using the IncuCyte Zoom system. D, E-cadherin expression does not change invasion in 143B cells. E, Using mRNA expression of CDH1 (E-cadherin), empirical probably density functions of EMT scores for CDH1-high (red) and CDH1-low (blue) TCGA sarcoma sample were constructed by interpolation of the EMT score histogram (E < 0.5, 0.5 ≤ E/M ≤ 1.5, M > 1.5). EMT score distribution showed no significant difference when separated for E-cadherinhigh versus E-cadherinlow expression from analysis of publicly available sarcoma datasets.
- Figure 3.
E-cadherin inhibits anchorage-independent growth of sarcomas. A, Anchorage-independent growth of 143B cells expressing E-cadherin was significantly inhibited. B and C, E-cadherin expression leads to reduces spheroid size in 143B (B) and U2OS cells (C).
- Figure 4.
Ectopic expression of E-cadherin in sarcoma cells inhibits phospho-CREB levels. A, A phospho-kinase array revealed that E-cadherin led to downregulation of phospho-CREB. B and C, E-cadherin-mediated phospho-CREB inhibition was verified by ELISAs (B) and Western blotting (C). D, Abrams canine osteosarcoma cells exhibited reduced phospho-CREB in E-cadherin-overexpressing cells. E, qRT-PCR confirmed knockdown of CREB with two independent siRNAs. F, Western blotting to confirm knockdown of CREB in 143B cells. G, CREB knockdown led to a modest downregulation of 143B colony growth in soft agar.
- Figure 5.
TBX2 knockdown phenocopies E-cadherin–mediated CREB inhibition. A and B, TBX2 mRNA is downregulated in E-cadherin–expressing 143B cells (A) and U2OS cells (B). C, CREB knockdown with two independent siRNAs had no effect on TBX2 mRNA. D–F, Conversely, TBX2 knockdown, verified in D led to a significant reduction in CREB mRNA (E) and protein level (F).
- Figure 6.
E-cad/TBX2 interplay mediates anchorage-independent growth. A, Intracellular regulatory circuit modulating anchorage-independent (A.I.) growth. B, Mathematical model predicts similar effects of Ecad-OE and TBX2-KD on anchorage-independent growth. C, TBX2 knockdown using two independent siRNAs inhibited anchorage-independent growth of 143B cells. D, TBX2 downregulation did not affect sphere formation in the presence of ectopic E-cadherin expression. E, TBX2 knockdown upregulates E-cadherin mRNA. F, Western blot analysis of U2OS cells upon TBX2 knockdown indicates E-cadherin protein is not upregulated by loss of TBX2. Prostate cancer (PC) cell line, LNCaP, was used as a positive control for E-cadherin expression. G, The E-cadherin promoter is differentially methylated in sarcomas as compared with carcinomas. H, Synovial sarcomas, which often display epithelioid histopathologic features, have the lowest levels of E-cadherin promoter methylation of all sarcoma subtypes. I, Among soft-tissue sarcoma histologic subtypes, E-cadherin mRNA expression is the highest in synovial sarcomas. Letters indicate statistically significant associations between groups. Undifferentiated pleomorphic sarcoma (UPS), liposarcoma (LPS), leiomyosarcoma (LMS), synovial sarcoma (SS), malignant peripheral nerve sheath tumors (MPNST), and myxofibrosarcoma (MFS).
- Figure 7.
E-cadherin inhibits spheroid formation by increased cell–cell adhesion. A, E-cadherin causes tighter clustering of cells during spheroid formation. B, A mechanical model illustrates the relationship between particle distance to adhesion. C, A mechanical model predicts E-cadherin drives down spheroid size through an increase in cell–cell adhesion. D and E, As predicted in the model, ectopic E-cadherin expression increases cell–cell adhesion in 143B (D) and U2OS (E) cells.
Additional Files
Supplementary Data
- Figure S1 - S1. E-cadherin over-expression does not alter sarcoma cell growth in monolayer culture.
- Figure S2 - S2. Ectopic E-cadherin expression in sarcoma cells does not alter EMT.
- Figure S3 - S3. CREB knockdown indicates it is downstream of E-cadherin and TBX2.
- Figure S4 - S4. E-cadherin induces E- to N-cadherin switching in sarcoma cells.
- Figure S5 - S5. Revised E-cad/TBX2 signaling model.
- Figure S6 - S6. A mechanical model relates cell-cell adhesion to spheroid size.
- Supplementary Data - Supplementary Data
Volume 17, Issue 6
