CEACAM1 controls the EMT switch in murine mammary carcinoma in vitro and in vivo.

We analyzed the molecular basis for carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1)-controlled inhibition of epithelial-mesenchymal transition (EMT) in a mouse model for mammary adenocarcinoma (WAP-T mice). We demonstrate that silencing of CEACAM1 in WAP-T tumor-derived G-2 cells induces epithelial-mesenchymal plasticity (EMP), as evidenced by typical changes of gene expression, morphology and increased invasion. In contrast, reintroduction of CEACAM1 into G-2 cells reversed up-regulation of genes imposing mesenchymal transition, as well as cellular invasion. We identified the Wnt-pathway as target for CEACAM1-mediated repression of EMT. Importantly, ß-catenin phosphorylation status and transcriptional activity strongly depend on CEACAM1 expression: CEACAM1high G-2 cells displayed enhanced phosphorylation of ß-catenin at S33/S37/T41 and decreased phosphorylation at Y86, thereby inhibiting canonical Wnt/ß-catenin signaling. We identified Src-homology 2 domain-containing phosphatase 2 (SHP-2) as a critical binding partner of CEACAM1 that could modulate ß-catenin Y86 phosphorylation. Hence, CEACAM1 serves as a scaffold that controls membrane proximal ß-catenin signaling. In vivo, mammary tumors of WAP-T/CEACAM1null mice displayed increased nuclear translocation of ß-catenin and a dramatically enhanced metastasis rate compared to WAP-T mice. Hence, CEACAM1 controls EMT in vitro and in vivo by site-specific regulation of ß-catenin phosphorylation. Survival analyses of human mammary carcinoma patients corroborated these data, indicating that CEACAM1 is a prognostic marker for breast cancer survival.

Loss of Sox9 function results in defective chondrocyte differentiation of mouse embryonic stem cells in vitro.

The transcription factor Sox9 plays an important role during chondrogenesis. After early conditional inactivation of Sox9 in mesenchymal limb bud cells of mice, mesenchymal condensations as well as cartilage and bone are completely absent in the developing limbs. We analyzed chondrogenic differentiation of Sox9-/- mouse embryonic stem cells in vitro, using two clones with different targeted mutations. We found that the development of mature and hypertrophic chondrocytes is completely inhibited in the absence of Sox9 confirming that Sox9 is required for the formation of cartilage. In contrast, Sox9+/- mouse embryonic stem cells showed continuous but reduced differentiation into mature chondrocytes. Interestingly, the formation of early chondrogenic condensations expressing characteristic marker genes such as scleraxis, Sox5 and Sox6 was not inhibited in the absence of Sox9 in vitro. Thus, we propose that the earliest step of chondrogenesis could be regulated by a non cell-autonomous function of Sox9.