(F to H) Liver organ (F) or lung (G) metastasis obtained from Balb/c mice after mammary fat pad injections (= 6 mice per group) with 4T1-Luc cells stably expressing Scr shRNAs or CerS4-shRNAs in response to transfections using vector and Smad7 or Scr shRNA and IFT88-shRNAs for inhibition of cilia formation was measured ex vivo using chemiluminescence. inhibitory factor Smad7, which limited the trafficking of TRI/II to primary cilia. Expression of a mutant TRI that signals but does not interact with Smad7 prevented the CerS4-mediated inhibition of migration in various cancer cells. Genetic deletion or knockdown of CerS4 prevented the formation of the Smad7-TRI inhibitory complex and increased the association between TRI and the transporter Arl6 through a previously unknown cilia-targeting signal (Ala31Thr32Ala33Leu34Gln35) in TRI. Mutating the cilia-targeting signal abolished the trafficking of TRI to the primary cilia. Localization of TRI to primary cilia activated a Vacquinol-1 key mediator of Shh signaling, Smoothened (Smo), which stimulated cellular migration and invasion. TRI-Smo cross-talk at the cilia in CerS4-deficient 4T1 mammary cancer cells induced liver metastasis from orthotopic allografts in both wild-type and CerS4-deficient mice, which was prevented by overexpression of Smad7 or knockdown of intraflagellar transport protein 88 (IFT88). Overall, these data reveal a ceramide-dependent mechanism that suppresses cell migration and invasion by restricting TRI/II-Shh signaling selectively at the plasma membrane of the primary cilium. INTRODUCTION Transforming growth factorC (TGF-) signaling is involved in the regulation Vacquinol-1 of various cellular signaling processes, including apoptosis, cell proliferation, differentiation, and migration (1C4). TGF- signaling is activated by the binding of the ligand to its specific serine-threonine kinase TGF- type I and type II receptors (TRI/II) on the plasma membrane (PM) (1C4). The ligand binding initiates the formation of the TRI/II heteromeric complex, in which TRII phosphorylates and activates TRI (1C4). Activation of Vacquinol-1 the TRI leads to the recruitment and formation of Smad protein complexes, which are translocated to the nucleus for the regulation of target genes (5C8). Inhibitory Smad7 negatively regulates TGF- signaling by binding TRI, leading to the recruitment of Smurf2, an E3 ubiquitin ligase that labels the TRI-Smad7 complex for degradation (9C13). The primary cilium is an organelle with a distinct membrane composition of lipids and proteins, which controls various signaling functions, such as enhanced cell-to-cell communication, autophagy, and/or cell migration (14C16). Intraflagellar transport (IFT) is a cargo-trafficking pathway, involved in cilium genesis, which maintains the microtubule axoneme (16C18). IFT machinery along with several proteins encoded by genes mutated in Bardet-Biedl syndrome (BBS) provides specificity for ciliary cargo transport (16C18). This includes targeting several receptors, including G proteinCcoupled receptors, to cilia via binding of BBS, such as BBS3 (Bardet-Biedl syndrome 3 protein) [Arl6 (adenosine diphosphateCribosylation factor-like protein 6)], to their cilia transport signal (CTS) comprising AX(S/A)XQ sequence (X is any amino acid) (17, 18). Sonic hedgehog (Shh) signaling is localized to primary cilia with a complex inhibitory (Patched) and activating [Smoothened (Smo)] pathways (19C21), leading to increased cell migration and metastasis. TRI/II signaling has been observed at the base of primary cilia (22), and ciliary TGF- signaling is linked to enhanced cell migration (23, 24). Ceramide, a bioactive signaling sphingolipid, is involved in the regulation of stress-related antiproliferative Vacquinol-1 responses in cancer cells, such as apoptosis, mitophagy, and/or necroptosis (25). Endogenous ceramides are synthesized de novo by six distinct ceramide synthases, CerS1 to CerS6 (26C29), which are specialized for the synthesis of ceramides with different fatty acyl chain lengths. For example, CerS5/CerS6 induces medium-chain C12- to C16-ceramides, CerS1/CerS4 induces long-chain C18- to C20-ceramides, and CerS2 Rabbit Polyclonal to SSXT induces very-long-chain C22- to C24-ceramides (26C29). CerS3, which is expressed selectively in testes and skin tissues, generates ultralong-chain ceramides (30, 31). Vacquinol-1 Ceramides with different fatty acyl chain lengths play distinct physiological roles in various biological processes, including providing skin barrier, liver homeostasis, insulin resistance, induction of apoptosis, and regulation of cancer pathogenesis (32C39). However, the roles of ceramides generated by CerS enzymes in the regulation of cancer cell migration and/or metastasis.
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