Explore the Regulatory Network of AKT signaling in Neural Crest Development and Cancer Biology

Abstract

DDX3X syndrome is a genetic disease caused by mutations in RNA helicase DDX3X with developmental delay and intellectual disability (ID). As we noticed that many patients also exhibit neural crest (NC) related defects, including craniofacial disorders and congenital heart disease, some of the prevalent birth defects, we hypothesized that DDX3 plays a role in NC development. Using the amphibian model, Xenopus tropicalis, we show that DDX3 induces NC formation through AKT activity in a helicase-dependent manner. DDX3 KD reduces AKT activity, leading to the destabilization of β-catenin, a downstream target of AKT and canonical Wnt signaling. The regulation of DDX3 on AKT activity in NC induction is likely mediated by RAC1, whose translation is facilitated by the RNA helicase activity of DDX3. Similar to DDX3X, mutations in RAC1 and AKT were also shown to cause ID and NC-related defects in humans, suggesting their function in NC development. We show that depletion of RAC1 or AKT2 impairs expression of NC marker snai2, and disrupts normal craniofacial morphogenesis. These results indicate that the function of DDX3 in modulating NC development through AKT activity is conserved between frogs and humans, advancing our understanding of the pathology of the NC-related birth defects caused by mutations in DDX3X, RAC1 and AKT. As an X chromosome-linked genetic disease, DDX3X syndrome affects considerably more female patients but the underpinning mechanisms of this sexual dimorphism remain largely unclear. It has been speculated that males with inactivating mutations in DDX3X suffer from embryonic lethality. However, recent studies suggested that DDX3X depletion might trigger compensatory expression of DDX3Y, a close homolog on the Y chromosome. We therefore hypothesized that DDX3X inhibits DDX3Y expression, and show that loss of DDX3X increases the DDX3Y expression through divergent mechanisms in two different human cell lines. In the glioma cell line U87-MG, a novel protein level regulation of DDX3Y by DDX3X was revealed, while in the colon cancer cell line HCT116, the regulation is more predominantly on the mRNA level of DDX3Y. These results provide a potential mechanistic explanation for the compensatory expression of DDX3Y induced by loss of DDX3X. The disintegrin metalloproteinases (ADAMs) are key regulators of cell signaling by cleaving cell-surface signaling molecules. ADAM9 has been implicated in several human diseases. In particular, ADAM9 is highly expressed in nearly all types of solid tumors, and the expression often correlates with poor prognosis. Understanding the downstream signaling pathways modulated by ADAM9 will provide important insight into its functions in carcinogenesis and tumor progression. Here we apply a systematic and non-biased approach, integrating the transcriptomics and the cellular proteomics analysis, to identify the downstream targets of ADAM9. As we reported previously that AKT is a downstream target of ADAM9, we show that with ADAM9 depletion, the FoxO signaling, one of the major pathways downstream of AKT, is found to be enriched in the RNA-seq analysis, while the transcription factor FoxO3 is upregulated in protein level. These analyses suggest that FoxO signaling is activated with ADAM9 KD, which is validated by western blotting and RT-qPCR. These results indicate that ADAM9 represses the activation of FoxO signaling, a tumor suppressor pathway, likely through activating AKT, providing a potential mechanism for the oncogenic roles of ADAM9 in various cancer types.