Microarray analysis revealed genes of the posterior HOXD locus normally involved in bone formation to be over-expressed in primary Ewing sarcoma (ES). The expression of posterior HOXD genes was not influenced via ES pathognomonic EWS/ETS translocations. However, knock down of the dickkopf WNT signaling pathway inhibitor 2 (DKK2) resulted in a significant suppression of HOXD10, HOXD11 and HOXD13 while over-expression of DKK2 and stimulation with factors of the WNT signaling pathway such as WNT3a, WNT5a or WNT11 increased their expression. RNA interference demonstrated that individual HOXD genes promoted chondrogenic differentiation potential, and enhanced expression of the bone-associated gene RUNX2. Furthermore, HOXD genes increased the level of the osteoblast- and osteoclast-specific genes, osteocalcin (BGLAP) and platelet-derived growth factor beta polypeptide (PDGFB), and may further regulate endochondral bone development via induction of parathyroid hormone-like hormone (PTHLH). Additionally, HOXD11 and HOXD13 promoted contact independent growth of ES, while in vitro invasiveness of ES lines was enhanced by all 3 HOXD genes investigated and seemed mediated via matrix metallopeptidase 1 (MMP1). Consequently, knock down of HOXD11 or HOXD13 significantly suppressed lung metastasis in a xeno-transplant model in immune deficient mice, providing overall evidence that posterior HOXD genes promote clonogenicity and metastatic potential of ES.

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Ewing sarcoma (ES), an osteogenic malignancy that mainly affects children and young adults, is characterized by early metastasis to lung and bone. Microarray analysis revealed genes of the posterior HoxD locus to be clearly over-expressed in primary ES. Especially, genes such as HOXD10, HOXD11 and HOXD13 normally involved in bone formation and ossification pattern of bones are significantly over-expressed in ES. The expression of HOXD10, HOXD11 or HOXD13 seems not to be influenced via ES pathognomonic ews/ets translocations, since RNA interference of EWS-FLI1 or its over-expression in mesenchymal stem cells did not influence posterior HOXD expression. However, RNA knock down of the dickkopf 2 homolog DKK2, a canonical WNT/beta-catenin agonist and critical mediator of osteolytic tumor growth in ES (Hauer et al., 2013) resulted in a significant suppression of HOXD11 and HOXD13 expression. Subsequently, RNA interference of individual HoxD genes demonstrated posterior HOXD genes to suppress neuronal differentiation potential of ES. Furthermore, HOXD10, HOXD11 and HOXD13 were found to be critical for chondrogenic, however not for osteogenic differentiation, but promote the expression of bone-associated genes such as RUNX2. HOXD11 and HOXD13 further promote in vitro proliferation and contact independent growth of ES. Similarly, in vitro invasiveness of ES lines was dependent on HOXD10, HOXD11 as well as HOXD13 and seemed mediated via Matrix Metalloproteinase 1 (MMP1), since down-regulation of individual HoxD genes likewise resulted in a suppression of MMP1 expression. In line with these observations, knock down of HOXD11 or HOXD13 significantly suppressed metastasis in a xeno-transplant model in immune deficient Rag2-/-gammaC-/- mice. These data overall provide evidence that posterior HOXD genes are critical mediators of the differentiation phenotype of ES concurrently promoting their metastasis potential. Reference: Hauer K, Julia Calzada-Wack J, Steiger K, Grunewald TG, Baumhoer D, Plehm S, Buch T, Prazeres da Costa O, Esposito I, Burdach S, Richter GHS (2013). DKK2 mediates osteolysis, invasiveness and metastatic spread in Ewing sarcoma. Cancer Res. 73(2): 967-77. Citation Format: Kristina von Heyking, Laura Roth, Miriam Ertl, Stefan Burdach, Günther H. Richter. The HoxD locus: Its contribution to the osteogenic phenotype and malignancy of Ewing sarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3106. doi:10.1158/1538-7445.AM2014-3106

Ewing sarcoma (ES), an osteogenic malignancy that mainly affects children and young adults, is characterized by early metastasis to lung and bone. Microarray analysis revealed genes of the HoxD locus to be clearly over-expressed in primary ES. Especially, genes such as HOXD10, HOXD11 and HOXD13 normally involved in bone formation and ossification pattern of bones are significantly over-expressed in ES. The expression of HOXD10, HOXD11 or HOXD13 seems not to be influenced via ES pathognomonic ews/ets translocations, since RNA interference of EWS-FLI1 or its over-expression in mesenchymal stem cells did not influence HOXD expression. However, RNA knock down of DKK2, a canonical WNT/β-catenin agonist and critical mediator of osteolytic tumor growth in ES resulted in a significant suppression of HOXD11 and HOXD13 expression. Subsequently, RNA interference of individual HoxD genes demonstrated HOXD11 and HOXD13 to be critical for in vitro proliferation and contact independent growth of ES. Similarly, in vitro invasiveness of ES lines was dependent on HOXD10, HOXD11 as well as HOXD13 and seemed mediated via MMP1, since down-regulation of individual HoxD genes similarly resulted in a suppression of MMP1 expression. But, HoxD genes only marginally influenced the expression of osteogenic genes in ES nor did they promote its chondrogenic or osteogenic differentiation. Whether HOXD10, HOXD11 or HOXD13 expression further metastasis or contribute to osteolytic tumor growth is currently investigated in a xenograft model using Rag2-/-gammaC-/- mice and data will be presented. Citation Format: Guenther HS Richter, Kristina von Heyking, Laura Roth, Miriam Ertl, Stefan Burdach. The HoxD locus: Its contribution to osteogenic phenotype and malignancy of Ewing sarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A51.