β-catenin is a node at orchestrating distinct aspects of epithelial-mesenchymal transition (EMT) and malignant mammary tumor progression

Canonical Wnt signaling plays crucial roles in the progression of many cancer types. In canonical Wnt signaling, β-catenin acts as a controller determining the transcriptional output: via its N-terminus it recruits the signaling coactivators Bcl9 and Bcl9/9l and via the C-terminus it interacts with the general transcriptional machinery. In the intestine, the C terminal output is essential, while the N-terminus controls only a subset of target genes. In breast cancer, the relative contribution of b-catenin’s different output is not known. Also not known is the functional contribution of β-catenin’s a role in cadherin-mediated cell adhesion, a function which confounds the analysis of conventional loss of function models. To address these unknowns, we combined the MMTV-PyMT mouse model of metastatic breast cancer with mouse lines carrying mutations in β-catenin that abolish its function completely or specifically target the N or C-terminal transcriptional outputs. Notably, the complete lack of β-catenin resulted in apoptosis of mammary tumor cells in vivo and in vitro. In contrast, the loss of β-catenin’s transcriptional functions did not provoke apoptosis but did diminish cell proliferation, epithelial-mesenchymal transition (EMT) and cell migration in vitro. This was also reflected in a reduction in primary tumor growth, tumor invasion, and metastasis formation in vivo. Whole transcriptome analysis identified subsets of genes, which were specifically regulated either by β-catenin’s N or C-terminal activities. Intriguingly, the N-terminal output was critical for TGFb-induced EMT and metastasis formation. The observations from the mouse models correlated with expression studies in human breast cancers. RNA-Seq of β-cateninfl/fl ( WT) and β-cateninD164A/-,β-cateninΔC/- and β-catenindm/- mutant cell lines on treatment with TGFb or Wnt3a was performed in biological duplicates

经典Wnt信号通路（Canonical Wnt signaling）在多种癌症的进展过程中发挥关键作用。在该通路中，β-连环蛋白（β-catenin）作为调控因子决定转录输出：其N端可招募信号转导辅激活因子Bcl9与Bcl9/9l，C端则与通用转录机器相互作用。在肠道组织中，β-连环蛋白的C端转录输出至关重要，而N端仅调控部分靶基因。在乳腺癌中，β-连环蛋白不同转录输出的相对贡献尚不明确；此外，β-连环蛋白在钙粘蛋白介导的细胞黏附中的功能贡献也尚未明晰，这一功能会干扰常规功能缺失模型的分析。为解决上述未知问题，本研究将转移性乳腺癌的MMTV-PyMT小鼠模型与携带β-连环蛋白突变的小鼠品系相结合，这些突变可完全废除β-连环蛋白的功能，或特异性靶向其N端、C端转录输出功能。值得注意的是，在体内与体外环境中，β-连环蛋白的完全缺失会导致乳腺肿瘤细胞发生凋亡。与之相反，β-连环蛋白转录功能的缺失并不会引发细胞凋亡，但会在体外降低细胞增殖、上皮间质转化（epithelial-mesenchymal transition, EMT）与细胞迁移能力。该结果同样体现在体内原发肿瘤生长、肿瘤侵袭与转移形成的减弱上。全转录组分析鉴定出了分别受β-连环蛋白N端或C端活性特异性调控的基因子集。有趣的是，N端转录输出对于转化生长因子β（TGFβ）诱导的上皮间质转化与转移形成至关重要。小鼠模型的观测结果与人类乳腺癌的表达谱研究结果相一致。本研究对经转化生长因子β（TGFβ）或Wnt3a处理的β-连环蛋白fl/fl（野生型，WT）、β-连环蛋白Δ164A/-、β-连环蛋白ΔC/-及β-连环蛋白dm/-突变细胞系开展了转录组测序（RNA-Seq）实验，设置了两个生物学重复。