Functional loss of the cell-cell adhesion molecule E-cadherin is an essential event for epithelial-mesenchymal transition (EMT), a process that allows cell migration during embryonic development and tumour invasion. In most carcinomas, transcriptional repression has emerged as the main mechanism responsible for E-cadherin downregulation. Here, we report the identification of class I bHLH factor E2-2 (TCF4/ITF2) as a new EMT regulator. Both isoforms of E2-2 (E2-2A and E2-2B) induce a full EMT when overexpressed in MDCK cells but without affecting the tumorigenic properties of parental cells, in contrast to other EMT inducers, such as Snail1 or class I bHLH E47. E-cadherin repression mediated by E2-2 is indirect and independent of proximal E-boxes of the promoter. Knockdown studies indicate that E2-2 expression is dispensable for maintenance of the EMT driven by Snail1 and E47. Comparative gene-profiling analysis reveals that E2-2 factors induce similar, yet distinct, genetic programs to that induced by E47 in MDCK cells. These results, together with the embryonic expression pattern of Tcf4 and E2A (which encodes E12/E47), support a distinct role for E2-2 and suggest an interesting interplay between E-cadherin repressors in the regulation of physiological and pathological EMT processes. Supplementary material available online at

Accumulating evidence points to reactive oxygen species (ROS) as important signaling molecules for cardiomyocyte differentiation in embryonic stem (ES) cells. Given that ES cells are normally maintained and differentiated in medium containing supraphysiological levels of glucose (25 mM), a condition which is known to result in enhanced cellular ROS formation, we questioned whether this high glucose concentration was necessary for cardiomyocyte lineage potential. We show here that ES cells cultured in physiological glucose (5 mM), maintained their general stemness qualities but displayed an altered mitochondrial metabolism, which resulted in decreased ROS production. Furthermore, ES and induced pluripotent stem (iPS) cells differentiated in lower glucose concentrations failed to generate cardiomyocyte structures; an effect mimicked with antioxidant treatments using catalase, N-acetyl cysteine and mitoubiquinone, under high glucose conditions in ES cells. Molecular analysis revealed that ES cells differentiated in 5 mM glucose had reduced expression of the pro-cardiac NOX4 gene and diminished phosphorylation of p38 mitogen-activated protein kinase (MAPK), together with specific changes in the cardiac transcriptional network. These outcomes could be reversed by supplementation of low glucose cultures with ascorbic acid, paradoxically acting as a pro-oxidant. Furthermore, forced expression of an upstream p38 MAPK kinase (MKK6) could bypass the requirement for ROS during differentiation to cardiomyocytes under low glucose conditions, illustrating a key role for p38 in the cardiac differentiation program. Together these data demonstrate that endogenous ROS control is important for cardiomyocyte formation from ES cells, and furthermore that supraphysiological glucose, by supplying ROS, is absolutely required.

Inherited KIF1B loss-of-function mutations in neuroblastomas and pheochromocytomas implicate the kinesin KIF1B as a 1p36.2 tumor suppressor. However, the mechanism of tumor suppression is unknown. We found that KIF1B isoform β (KIF1Bβ) interacts with RNA helicase A (DHX9), causing nuclear accumulation of DHX9, followed by subsequent induction of the proapoptotic XIAP-associated factor 1 (XAF1) and, consequently, apoptosis. Pheochromocytoma and neuroblastoma arise from neural crest progenitors that compete for growth factors such as nerve growth factor (NGF) during development. KIF1Bβ is required for developmental apoptosis induced by competition for NGF. We show that DHX9 is induced by and required for apoptosis stimulated by NGF deprivation. Moreover, neuroblastomas with chromosomal deletion of 1p36 exhibit loss of KIF1Bβ expression and impaired DHX9 nuclear localization, implicating the loss of DHX9 nuclear activity in neuroblastoma pathogenesis. SIGNIFICANCE:KIF1Bβ has neuroblastoma tumor-suppressor properties and promotes and requires nuclear-localized DHX9 for its apoptotic function by activating XAF1 expression. Loss of KIF1Bβ alters subcellular localization of DHX9 and diminishes NGF dependence of sympathetic neurons, leading to reduced culling of neural progenitors, and, therefore, might predispose to tumor formation. Cancer Discov; 4(4); 434-51.