A Novel Signaling Pathway that Governs Tumor Metastasis: Ceramide Regulates Direct Crosstalk Between TGF‐ß and Sonic Hedgehog Signaling

In the past twenty years, specific sphingolipids have become recognized for their participation in membrane functions and signaling events that control a wide array of cellular processes. Ceramide is considered as a main molecule of bioactive sphingolipid metabolites, are involved in signaling pathways that regulate cell proliferation, apoptosis, motility, differentiation, and angiogenesis. More recent studies have introduced bioactive sphingolipids and their metabolic enzymes take a role as regulated component in migration and cell mobility processes. However, the molecular mechanism of ceramide involved is unknown. Here, we investigated the effect of ceramide synthases (CerS) and ceramide on migration and its related signal pathways in situ and in vivo model. Interestingly, our data show that among CerS only CerS4/ceramide is involved to cell migration and tumor metastasis. Here, we also have generated CerS4−/− mice for in vivo studies. Excitingly, we observed that genetically loss of CerS4 resulted in severe irreversible alopecia, which was associated with hyper‐proliferation and migration of keratinocytes. Mechanistically, we show here that genetic loss or shRNA‐mediated knockdown of CerS4 enhances cell migration by which ligand‐independent signaling of TGF‐beta receptors I and II (TßRI/II) in various cell types, including keratinocytes, mouse embryonic fibroblasts and cancer cells. Mechanisms that regulate membrane localization of TßRI/II for the regulation of cell migration and metastasis are largely unknown. We identified that alterations in ceramide metabolism by molecular knockdown and/or genetic deletion of CerS4, reducing bioactive C 18 ‐C 20 ‐ceramides, results in enhanced TßRI‐II signaling at the plasma membrane by inhibiting Smad7 recruitment to the receptor, leading to increased cell migration/invasion via activation of the sonic hedgehog (Shh) pathway. Mechanistically, inhibition of Smad7‐TßRI/II interaction in response to CerS4 knockdown led to the activation of the Shh signaling via enhanced association/cross‐talk between TßRI‐II and smoothened (Smo), a signaling Shh receptor, at the plasma membrane. In reciprocal studies, reconstitution of CerS4/ceramide inhibited TGF‐ß‐Shh crosstalk, reducing cancer cell migration and inhibition of the cancer cell colonization/lung metastasis in SCID mice. Moreover, knockdown of CerS4 in cancer cells, but not systemic deletion of CerS4 in mice, increased metastasis of breast cancer cells from mammary pads to the liver, which was inhibited by Smad7 expression or Smo knockdown. Thus, these data reveal that CerS4/ceramide is a novel factor that regulates the recruitment of Smad7 to the TßRI, controlling the stability/surface expression of the receptor, which modulates TßRI/II‐Smo crosstalk, controlling cell migration and cancer metastasis. Support or Funding Information 1R01 DE016572