TGF-β receptor I/II trafficking and signaling at primary cilia are inhibited by ceramide to attenuate cell migration and tumor metastasis

Abstract

Signaling by the transforming growth factor–β (TGF-β) receptors I and II (TβRI/II) and the primary cilia-localized sonic hedgehog (Shh) pathway promote cell migration and, consequently, tumor metastasis. In contrast, the sphingolipid ceramide inhibits cell proliferation and tumor metastasis. We investigated whether ceramide metabolism inhibited TβRI/II trafficking to primary cilia to attenuate cross-talk between TβRI/II and the Shh pathway. We found that ceramide synthase 4 (CerS4)–generated ceramide stabilized the association between TβRI and the inhibitory factor Smad7, which limited the trafficking of TβRI/II to primary cilia. Expression of a mutant TβRI 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-TβRI inhibitory complex and increased the association between TβRI and the transporter Arl6 through a previously unknown cilia-targeting signal (Ala31Thr32Ala33Leu34Gln35) in TβRI. Mutating the cilia-targeting signal abolished the trafficking of TβRI to the primary cilia. Localization of TβRI to primary cilia activated a key mediator of Shh signaling, Smoothened (Smo), which stimulated cellular migration and invasion. TβRI-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 TβRI/II-Shh signaling selectively at the plasma membrane of the primary cilium.

IFT80 Is Required for Fracture Healing Through Controlling the Regulation of TGF-β Signaling in Chondrocyte Differentiation and Function

“…Primary cilium has been reported to mediate TGF‐β1–induced recruitment of human bone MSCs in a SMAD3‐dependent manner . A recent study revealed that mutation of the cilia‐targeting signal abolished the trafficking of TGF‐βRI to the primary cilia . In fibroblasts from the Tg737 orpk mutant mouse, where IFT88 is mutated, clathrin‐dependent endocytosis is disrupted at stunted primary cilia, and accumulation of TGF‐β receptors and activation of SMAD2/3 are reduced .…”

Balance between senescence and apoptosis is regulated by telomere damage–induced association between p16 and caspase-3

“…SA-ß-galactosidase activity was also measured for paw tissues of mice from indicated genotypes using the same kit. All animal experiment protocols were approved by the Institutional Animal Care and Use Committee at the Medical University of South Carolina (27,(54)(55)(56).…”

“…Tumor volume was measured every alternative day using digital calipers and treatments were performed using oral gavage method every day. At the end of the experiment the mice were euthanized and tumor tissues were collected (27,(54)(55)(56)(57).…”

“…Tumor xenograft experiments were analyzed using two-way ANOVA with Tukey's posthoc test and testes weights from G4 and G5 mice were analyzed using one-way ANOVA with Tukey's posthoc test. The results were analysed using GraphPad Prism 7 software and P<0.05 was considered statistically significant (27,(54)(55)(56) Arab, K., Park, Y. J., Lindroth, A. M., Schafer, A., Oakes, C., Weichenhan, D., Lukanova, A., Lundin, E., Risch, A., Meister, M., Dienemann, H., Dyckhoff, G., Herold-Mende, C., Grummt, I., Niehrs, C., and Plass, C. ShRNA-mediated knockdown of ISPHK2 mRNA in A549 xenograft-derived tumors was confirmed using qPCR after the tumor growth measurements were completed. Data are means ± SD from three independent experiments, analysed by student's t-test (n=3, *P=0.039, **P=0.014).…”

“…Sphingolipids in cells (1 x 10 6 ) were measured using mass spectrometry by lipidomics shared resource facility at the Medical University of South Carolina, as we described previously (54). The lipid abundance was normalized to inorganic phosphate (Pi), Ultra-structure analysis using TEM Formalin fixed shScr and shTCF21 tumors treated with ABC294640 or vehicle control were used.…”