Cadherin-11 contributes to the heterogenous and dynamic Wnt-Wnt-β-catenin pathway activation in Ewing sarcoma

Abstract

Ewing sarcoma is the second most common bone cancer in children, and while patients who present with metastatic disease at the time of diagnosis have a dismal prognosis. Ewing sarcoma tumors are driven by the fusion gene EWS/Fli1, and while these tumors are genetically homogenous, the transcriptional heterogeneity can lead to a variety of cellular processes including metastasis. In this study, we demonstrate that in Ewing sarcoma cells, the canonical Wnt/β-Catenin signaling pathway is heterogeneously activated in vitro and in vivo, correlating with hypoxia and EWS/Fli1 activity. Ewing sarcoma cells predominantly express β-Catenin on the cell membrane bound to CDH11, which can respond to exogenous Wnt ligands leading to the immediate activation of Wnt/β-Catenin signaling within a tumor. Knockdown of CDH11 leads to delayed and decreased response to exogenous Wnt ligand stimulation, and ultimately decreased metastatic propensity. Our findings strongly indicate that CDH11 is a key component of regulating Wnt//β-Catenin signaling heterogeneity within Ewing sarcoma tumors, and is a promising molecular target to alter Wnt//β-Catenin signaling in Ewing sarcoma patients.

Fig 1. β-catenin depletion leads to reduced clonogenicity, anchorage independent growth, and metastatic dissemination in Ewing sarcoma cells.

(A) β-catenin was stably knocked down by shRNA (KD#1 and KD#2), and was confirmed by immunoblotting. Control is shRNA targeting GFP (CTRL). (B) Effect of β-catenin shRNA-knockdown significantly decreased colony formation in clonogenic assays. Shown are quantification of colonies per wells, as well as representative wells. (C) In non-adherent soft agar colony formation assays, β-catenin knockdown led to a significant decrease in colony formation. (D) in vivo tail vein injection models demonstrate that β-catenin knockdown leads to less metastatic dissemination to the liver as well as lungs. Quantifications represent mean and standard error of the mean from three or more experiments performed independently. Statistical significance was determined using a Mann-Whitney Test with multiple comparisons. ns, not significant, *p<0.05, **p<0.01, ****p<0.0001.

Fig 2. Wnt/β-catenin pathway activation is heterogenous within Ewing sarcoma tumors.

(A) Flowcytometry analysis of cultured Ewing sarcoma cell lines stably expressing the Wnt reporter 7TGC, demonstrate a small population of mCherry and eGFP expressing cells. (B) 7TGC expressing A4573 xenografts demonstrate a small subpopulation of mCherry and eGFP positive cells in vivo. The proportion of eGFP positive cells are significantly higher in vivo than in vitro. (C, D) Single cell RNA-seq of a A4573 xenograft demonstrates significant heterogeneity of Wnt pathway activation. (E) Tumor cells within A4573 xenografts demonstrate positive and negative correlation of various pathways with the IC-EwS signature. Fisher exact test was used to compare proportion of mCherry and eGFP positive cells in Fig 2B. ****p<0.0001.

Fig 3. β-catenin localizes in the cell membrane and cytoplasm in Ewing sarcoma cells.

(A) Immunohistochemistry confirms that β-catenin is highly localized in the cellular membrane. (B) Immunofluorescent staining demonstrates that β-catenin is highly expressed in the cytoplasm of Ewing sarcoma cells. (C) Western blot analysis of cellular fractions of ES cells demonstrate that β-catenin is abundantly expressed in the membranous fraction (Mem), and cytoplasmic fraction (Cyto), and minimally in the nuclear fraction (Nuc). Whole cell lysate (WCL) was loaded as control. Relative intensity of β-Catenin band measured by densitometry shown.

Fig 4. CDH11 is highly expressed by Ewing Sarcoma cells, and is bound to β-catenin.

(A) Ewing sarcoma cells were assessed for Cadherin expression, demonstrating strong CDH11 expression, while E-Cadherin and N-Cadherin is not expressed. Relative intensity of CDH11 band measured by densitometry shown. (B) Immunofluorescence of Ewing sarcoma cell lines demonstrates that CDH11 and β-catenin co-localize on the cytoplasmic portion of TC71 and A4573 cells. (C) Co-Immunoprecipitation shows that CDH11 is bound to β-catenin in Ewing sarcoma cells, while IgG input control (IgG IP) does not detect β-catenin.

Fig 5. Stable β-catenin expression, and Wnt ligand response is dependent on CDH11.

(A) β-catenin knockdown in Ewing sarcoma cells do not lead to alteration of CDH11 expression. (B) CDH11 knockout leads to loss of β-catenin expression. Flowcytometry confirms successful CDH11 knockout. Relative intensity of β-Catenin and CDH11 bands measured by densitometry shown. (C) 7TGC expressing Ewing sarcoma cells were exposed for six hours to supernatant from L cells expressing a Wnt3a overexpression vector (Wnt 6hr), wild type L cells as a control (L cell 6 hour), or regular culture media (Wnt(-)). Compared to control cells transfected with empty control vectors (Control), CDH11 knockdown cells have a delayed and diminished response to exogenous Wnt3a, measured by eGFP expression. Quantifications represent mean and standard error of the mean from three or more experiments performed independently. Statistical significance was determined using a Mann-Whitney Test with multiple comparisons. Fisher exact test was also used to compare proportion of mCherry and eGFP positive cells in Fig 5C. ns, not significant, ****p<0.0001.

Fig 6. CDH11 depletion in ES cells lead to reduced clonogenicity, migration, and morphology.

(A) CDH11 knock-out leads to significant reduction in colony number in clonogenic assays. (B) In anchorage independent soft agar growth assays, CDH11 knockdown leads to a trend towards a decrease in colony growth. (C) CDH11 knock-out leads to a significant reduction in the percentage of cells exhibiting neurite outgrowth, leading to a significant decrease of the area of cells. Representative images as well as measurement of cell area is demonstrated. (D) CDH11 knockout results in significant reduction of migration measured by chemotaxis migration in A4573 cells. Quantifications represent mean and standard error of the mean from three or more experiments performed independently. Statistical significance was determined using an unpaired T-test and Mann-Whitney Test with multiple comparisons. ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Fig 7. CDH11 depletion leads to decreased Ewing sarcoma metastasis.

(A-B) CDH11 knockout cells develop less metastatic burden in the lung and liver, following tail vein injection. (C) Kaplan-Meier Survival curve of overall survival for control and CDH11 knockout cells. (D) immunofluorescence demonstrates sustained CDH11 knockout in outgrown tumors. Statistical significance was determined using a Mann-Whitney Test with multiple comparisons. ns, not significant, **p<0.01, ****p<0.0001.