Intratumoral bidirectional transitions between epithelial and mesenchymal cells in triple-negative breast cancer.

Epithelial-mesenchymal transition (EMT) and its reverse process, mesenchymal-epithelial transition MET, are crucial in several stages of cancer metastasis. Epithelial-mesenchymal transition allows cancer cells to move to proximal blood vessels for intravasation. However, because EMT and MET processes are dynamic, mesenchymal cancer cells are likely to undergo MET transiently and subsequently re-undergo EMT to restart the metastatic process. Therefore, spatiotemporally coordinated mutual regulation between EMT and MET could occur during metastasis. To elucidate such regulation, we chose HCC38, a human triple-negative breast cancer cell line, because HCC38 is composed of epithelial and mesenchymal populations at a fixed ratio even though mesenchymal cells proliferate significantly more slowly than epithelial cells. We purified epithelial and mesenchymal cells from Venus-labeled and unlabeled HCC38 cells and mixed them at various ratios to follow EMT and MET. Using this system, we found that the efficiency of EMT is approximately an order of magnitude higher than that of MET and that the two populations significantly enhance the transition of cells from the other population to their own. In addition, knockdown of Zinc finger E-box-binding homeobox 1 (ZEB1) or Zinc finger protein SNAI2 (SLUG) significantly suppressed EMT but promoted partial MET, indicating that ZEB1 and SLUG are crucial to EMT and MET. We also show that primary breast cancer cells underwent EMT that correlated with changes in expression profiles of genes determining EMT status and breast cancer subtype. These changes were very similar to those observed in EMT in HCC38 cells. Consequently, we propose HCC38 as a suitable model to analyze EMT-MET dynamics that could affect the development of triple-negative breast cancer.

TRAF6 maintains mammary stem cells and promotes pregnancy-induced mammary epithelial cell expansion.

Receptor activator of nuclear factor (NF)-κB (RANK) signaling promotes pregnancy-dependent epithelial cell differentiation and expansion for mammary gland development, which requires NF-κB pathway-dependent Cyclin D1 induction and inhibitor of DNA binding 2 (Id2) pathway-dependent anti-apoptotic gene induction. However, the roles of tumor necrosis factor receptor-associated factor 6 (TRAF6) remain unclear despite its requirement in RANK signaling. Here we show that TRAF6 is crucial for both mammary stem cell maintenance and pregnancy-induced epithelial cell expansion. TRAF6 deficiency impairs phosphoinositide 3-kinase (PI3K)/AKT and canonical NF-κB pathways, whereas noncanonical NF-κB signaling remains functional. Therefore, we propose that TRAF6 promotes cell proliferation by activating PI3K/AKT signaling to induce retinoblastoma phosphorylation in concert with noncanonical NF-κB pathway-dependent Cyclin D1 induction. Furthermore, TRAF6 inhibits apoptosis by activating canonical NF-κB signaling to induce anti-apoptotic genes with the Id2 pathway. Therefore, proper orchestration of TRAF6-dependent and -independent RANK signals likely establishes mammary gland formation.