Research ArticleCancer

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

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Science Signaling  24 Oct 2017:
Vol. 10, Issue 502, eaam7464
DOI: 10.1126/scisignal.aam7464
  • Fig. 1 CerS4 knockdown mediates cell migration.

    (A) Fibronectin-coated Boyden chamber assays measuring migration of A549 cells after being transfected with control (Scr) or CerS1- or CerS4-targeted siRNA. Scale bar, 100 µm. (B) Effect of siRNA-mediated CerS4 knockdown on A549 cell migration measured by the clearance of fluorescent microspheres on fibronectin-coated wells. The ability of cells to create phagokinetic nonfluorescent tracks was evaluated by fluorescence microscopy and quantified using National Institutes of Health Image software. (C) As described in (A), using two shRNAs against CerS4. Scale bar, 100 µm. (D) As in (B), using UM-SCC-22A cells transfected with CerS4-targeted or control shRNA. Data are means ± SD of n = 3 independent experiments. *P < 0.05 by Student’s t tests.

  • Fig. 2 CerS4 knockdown induces cell migration through increased TβRI/II membrane trafficking and signaling.

    (A) Cell surface abundance of TβRI/II measured by flow cytometry in A549 cells transfected with CerS4-shRNAs or Scr shRNAs using two different sets of anti-TβRI or anti-TβRII antibodies. (B) Effects of TβRI inhibition by SB431542 on migration of A549 cells transfected with CerS4-targeted compared to Scr shRNAs, as measured in fibronectin-coated Boyden chambers. (C) Boyden chamber assays using fibronectin-coated chambers assessing migration of 4T1 cells after expression of wild-type (WT) or D/N-mutant TβRI (confirmed with Western blot, below the graph) and CerS4-targeted or control siRNA in the presence of vehicle or TGF-β (5 ng/ml). OD, optical density. (D) Effects of shRNA-mediated knockdown of CerS4 on total and cell surface protein abundance of Smad7, TβRI, and TβRII measured by biotin-labeling followed by Western blotting. Actin and Na+/K+-ATPase were used as controls for total or cell surface, respectively. Graph shows the amount of each protein at the cell surface (quantified relative to total protein in the whole lysate) in CerS4-shRNA–transfected cells (gray bars) relative to that in control, Scr shRNA–transfected cells (black bars). (E) Western blotting for Smad7 in whole-cell lysates from 4T1 cells cotransfected with a control (vector) or FLAG-tagged Smad7 expression vector and Scr- or CerS4-targeting shRNA. (F) Effects of WT-Smad7 on migration in Boyden chambers in A549 cells expressing Scr shRNA or CerS4-shRNAs were measured. (G to I) Luciferase reporter assay (G) and Western blotting (H and I) to assess the effects of CerS4 knockdown by shRNA in 4T1 cells on Smad-dependent promoter activation (G) and the phosphorylation of Smad3 (H) and Smad2 (I). Cells were cultured in the presence or absence of exogenous TGF-β exposure (5 ng/ml), as indicated. Data are means ± SD (n = 3 independent experiments). Blots are representative of n ≥ 3. *P < 0.05 by a Student’s t test on log-transformed data. AU, arbitrary units; MBP, myelin basic protein; BE, binding element.

  • Fig. 3 Genetic CerS4 inhibition results in increased outer membrane localization of TβRII and signaling to induce migration in CerS4−/− MEFs and keratinocytes.

    (A) Outer membrane abundance of TβRII in MEFs and skin keratinocytes isolated from age-matched WT and CerS4−/− mice was measured by flow cytometry using the anti-TβRII antibody. Data are means ± SD (n = 3 independent experiments; *P < 0.05). P values were calculated using t test on log-transformed data. (B and C) Effects of WT-Smad7 on the migration of WT and CerS4−/− skin keratinocytes were measured using fibronectin-coated Boyden chambers (B) or single-cell migration track assay (C) compared to vector-transfected controls. Data are means ± SD (n = 3 independent experiments). KO, knockout. (D and E) Effects of reconstitution of CerS4 on the migration of WT and CerS4−/− skin keratinocytes were measured using fibronectin-coated Boyden chambers (D) or single-cell tracking assay (E) compared to vector-transfected controls. Data are means ± SD (n = 3 independent experiments) (*P < 0.05). (F) Effects of reconstitution of CerS4WT versus catalytically inactive CerS4H212A/H213A, unable to generate ceramide, on migration in A549 cells, stably expressing CerS4-shRNA or Scr shRNA, were measured in fibronectin-coated Boyden chambers. Vector-transfected cells were used as controls. Data are means ± SD (n = 3 independent experiments). (G) Tet-induced CerS4-V5 at 24 to 72 hours in A549 cell extracts was detected by Western blotting using anti-CerS4 or anti-V5 antibodies. Actin was used as a loading control. Data represent at least three independent experiments. (H) Effects of TβRI/II cotransfections on A549 cell migration in the absence/presence (−/+tet) of CerS4 induction were measured in fibronectin-coated Boyden chambers. Vector-transfected A549 cells were used as controls. Data are means ± SD (n = 3 independent experiments). Student’s t test was performed.

  • Fig. 4 Association of TβRI with inhibitory Smad7 regulates receptor signaling.

    (A) Colocalization (white) of ceramide (Cer) (green), Smad7 (red), and TβRI (cyan) was detected by immunofluorescence in 4T1 cells stably expressing Scr shRNA or CerS4-shRNAs. Images represent three independent studies, which are quantified using ImageJ, and *P < 0.05 was considered significant. Scale bars, 100 µm. (B) Ceramides were measured using lipidomics and normalized to milligrams of protein. Data are means ± SD (n = 4 independent studies). (C) Ceramide-Smad7 interaction in immortalized MEFs isolated from WT or Cer4−/− mice (left) and in Scr shRNA– or CerS4-shRNA–transfected A549 cells (right) was measured by PLA using antibodies against ceramide and Smad7. PLA signals were quantified using the Duolink Image Tool. Data are means ± SD (n = 3 independent experiments; *P < 0.05). (D and E) TβRI-Smad7 (D) or Smad7-Smurf2 (E) association was determined by immunoprecipitation (IP) and Western blotting [immunoblotting (IB)] using antibodies that recognize TβRI (R1), Smad7, or Smurf2 in extracts isolated from A549 cells stably transfected with Scr shRNA or CerS4-shRNAs in the absence or presence of vector, CerS4H212A/H213A, or CerS4WT. Anti-IgG (IgG) antibody was used to detect nonspecific immunoprecipitation of proteins in these extracts. Actin was used as a loading control in Western blots. Blots shown represent at least three independent studies. (F) Association between TβRI (R1) and Smad7 was measured by PLA using antibodies that recognize TβRI or Smad7 in immortalized WT or CerS4−/− MEFs. Anti-IgG antibody was used as a negative control. PLA signals were quantified using the Duolink Image Tool. Data are means ± SD (n = 3 independent experiments; *P < 0.05). Scale bars, 100 µm. (G and H) Effects of Asn271Ala/Thr272Ala conversions in TβRI on Smad7 recognition (G) or on A549 cell migration or invasion (H) with/without CerS4 induction were measured using PLA or Matrigel-coated Boyden chamber invasion assay compared to vector- and WT-TβRI–transfected controls. Data are means ± SD (n = 3 independent experiments; *P < 0.05). Student’s t test was performed. EV, empty vector; RI, TβRI.

  • Fig. 5 Activation of TβRI/II signaling at the PM increases cell migration by Shh/Smo signaling in response to CerS4/ceramide knockdown.

    (A and B) Effects of shRNA-mediated CerS4 knockdown on Shh mRNA (A) or secreted protein abundance (B) were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) or enzyme-linked immunosorbent assay using Shh detection kit (Abcam). Scr shRNA–transfected A549 cells were used as controls. Data are means ± SD (n = 3 independent experiments; *P < 0.05). (C) Effects of Shh inhibition by robonikinin (+Rob) on A549 cell migration in response to siRNA-mediated CerS4 knockdown were measured using single-cell migration tracking assay compared to Scr-siRNA–transfected controls. Data are means ± SD (n = 3 independent experiments; *P < 0.05). (D and E) Effects of shRNA-mediated knockdown of Shh on the migration of A549 cells stably expressing CerS4-shRNA compared to Scr shRNA were measured in fibronectin-coated Boyden chambers (D). Quantification was performed using ImageJ (E). Data are means ± SD (n = 3 independent experiments; *P < 0.05). Scale bars, 100 µm. (F to H) Effects of Shh signaling inhibition by cyclopamine (10 μM) on Gli protein abundance (F), Gli mRNA (G), and cell migration (H) were measured in response to CerS4 knockdown compared to Scr shRNA–transfected controls. Quantification was performed using ImageJ (H, right). Data are means ± SD (n = 3 independent experiments; *P < 0.05). Scale bar, 100 µm. (I to K) Effects of Gli1 knockdown using siRNAs on Gli1 abundance (I), Gli1 mRNA (J), and cell migration (K) were measured in response to CerS4 knockdown compared to controls. Quantification was performed using ImageJ. Data are means ± SD (n = 3 independent experiments; *P < 0.05). Student’s t test was performed, and P values were calculated using t test on log-transformed data (for A, G, and J). Scale bar, 100 µm.

  • Fig. 6 Trafficking of TβRI/II to primary cilia membrane mediates the cross-talk between TβRI and Smo.

    (A) Effects of shRNA-mediated CerS4 knockdown on Smo-TβRI interaction were measured by immunoprecipitation using anti-TβRI antibody followed by Western blotting using anti-Smo or anti-TβRI antibodies compared to Scr-transfected 4T1 cells (bottom). Data are means ± SD (n = 3 independent experiments; *P < 0.05). Total protein abundance of Smo in cell extracts before immunoprecipitation studies was measured by Western blotting (top). (B) Interaction between TβRI-Smo and TβRII-Smo was measured by PLA using anti-TβRI, anti-TβRII, and anti-Smo antibodies in A549 cells expressing Scr shRNA or CerS4-shRNAs. PLA signals were quantified using the Duolink Image Tool. Data are means ± SD (n = 3 independent experiments; *P < 0.05). Scale bars, 100 µm. (C and D) TβRI and Smo interaction was measured by immunoprecipitation and Western blotting using anti-TβRI and anti-Smo antibodies in 4T1 (C) or A549 (D) cells stably expressing Scr shRNA or CerS4-shRNAs in response to vector or WT-Smad7 (top). Anti-IgG antibody was used as a negative control. Equal immunoprecipitation of TβRI was confirmed by Western blotting (middle). Actin was measured for equal input of proteins in ceramide binding studies. Data represent at least three independent studies. o/e, overexpression. (E) Effects of shRNA-mediated knockdown of CerS4 on the localization of TβRI in primary cilia membrane (black arrows) were measured in primary cilia-enriched fractions using EM with gold-labeled antibodies that recognize Ac-tubulin or TβRI compared to controls (Scr shRNA) (left and middle). EM images obtained using a gold-labeled anti–Glu-tubulin antibody in primary cilia-enriched fractions obtained from Scr shRNA– or CerS4-shRNA–transfected 4T1 cells were used as negative controls (right). Data represent at least three independent studies. Scale bars, 100 nm. (F) Primary cilia localization of TβRI was detected by colocalization of Ac-tubulin using immunofluorescence in response to CerS4 knockdown or genetic loss in A549, UM-SCC-22A (U22A), MEFs, and keratinocytes. Data represent at least three independent studies (n = 3). Scale bars, 100 µm. (G) Ceramide, TβRI, and Smo localization in primary cilium (merged yellow) was detected by immunofluorescence in 4T1 cells stably expressing Scr shRNAs or CerS4-shRNAs. Quantification was performed using ImageJ (bottom). Data are means ± SD (n = 3 independent experiments; *P <0.05). Scale bar, 100 µm. (H) Colocalization of Ac-tubulin (Ac-Tub) (green), TβRI (purple), and Smo (red) was measured by immunofluorescence using anti–Ac-Tub, anti-TβRI, and anti-Smo antibodies in 4T1 stably expressing Scr shRNAs or CerS4-shRNAs. Images represent three independent experiments, which are quantified using ImageJ. Data are means ± SD (n = 3 independent experiments; *P < 0.05) (bottom). Student’s t test was performed. Scale bars, 100 µm.

  • Fig. 7 TβRI/II trafficking to primary cilium by CTS and Arl6 in response to CerS4 knockdown in mammary cancer cells mediates liver metastasis.

    (A and B) Effects of shRNA-mediated knockdown of Arl6 and/or BBS4 on primary cilia localization of TβRI in PM/primary cilia by flow cytometry (A) and on cell migration or invasion in Boyden chambers (B) were measured in 4T1-Luc cells stably expressing shRNA against CerS4 (left). Data are means ± SD (n = 3 independent experiments; *P < 0.05). Knockdowns of Arl6 and BBS4 were confirmed by Western blotting (B, middle and right). Blots represent three independent studies. (C) Association between Arl6 and TβRI in 4T1 cells stably expressing Scr shRNAs or CerS4 shRNAs was measured by immunoprecipitation and Western blotting using anti-Arl6 and anti-TβRI antibodies in primary cilia-enriched fractions. Images were quantified using ImageJ. Data are means ± SD (n = 3 independent experiments; *P < 0.05). (D and E) Effects of WT-TβRI or mutant of TβRI (TβRImut) (Δ31-Ala-Thr-Ala-Leu-Gln-35) on its primary cilia localization or cell migration were measured by immunofluorescence (D) or fibronectin-coated Boyden chamber migration assay in the absence/presence of TGF-β (5 ng/ml) (E) compared to vector-transfected controls. Equal abundance of WT- and mutant-TβRI was confirmed by Western blotting (D, right). Data are means ± SD (n = 3 independent experiments; *P < 0.05). (F to H) Liver (F) or lung (G) metastasis obtained from Balb/c mice after mammary fat pad injections (n = 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. Data are means ± SD. Liver tumor nodules were detected and measured via hematoxylin and eosin staining by an independent pathologist (H). Data are means ± SD (n = 6; *P < 0.05). (I) Association of TβRI and Smo was detected by immunoprecipitation and Western blotting in liver tissues obtained from mice, as in (F). Data are means ± SD (*P < 0.05). Student’s t test and analysis of variance (ANOVA) with Tukey’s posttest for pairwise comparisons in animal/allograft studies in (F) and (G) were performed. P values were calculated using t test on log-transformed data (for A, C, and E). (J) Graphical summary. Our data suggest that CerS4-generated C18-/C20-ceramide forms an inhibitory ceramide-Smad7 complex, which inhibits the primary cilia membrane localization of TβRI/II and signaling, preventing the cross-talk between TβRI and Smo, attenuating Shh/Smo-dependent cell migration. Molecular and genetic inhibition of CerS4/ceramide prevents ceramide-Smad7 complex formation, inducing Arl6-dependent TβRI/II trafficking and signaling in primary cilia through novel CTS of the TβRI. Activated TβRI/II signaling in primary cilium by CerS4 and/or Smad7 knockdown induces TβRI-Smo interaction and Shh/Smo signaling, increasing cell migration and liver metastasis.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/502/eaam7464/DC1

    Fig. S1. CerS4 is down-regulated in most advanced metastatic tumor tissues.

    Fig. S2. CerS4 knockdown results in increased cell migration and invasion.

    Fig. S3. CerS4−/− mice exhibit irreversible alopecia and decreased C18- and/or C20-ceramide synthesis.

    Fig. S4. CerS4 induction increases C18- and C20-ceramide synthesis.

    Fig. S5. Analysis of Smad7 expression and the subcellular localization of TβRI/II after CerS4 knockdown.

    Fig. S6. CerS4 knockdown induces Shh abundance without affecting p-Smad3 abundance.

    Fig. S7. CerS4 knockdown enables cell migration through cross-talk between TβRI/II and Shh/Smo signaling.

    Fig. S8. Inhibition of primary cilia formation prevents CerS4 knockdown-induced cell migration and metastasis to the liver and lung.

  • Supplementary Materials for:

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

    Salih Gencer, Natalia Oleinik, Jisun Kim, Shanmugam Panneer Selvam, Ryan De Palma, Mohammed Dany, Rose Nganga, Raquela J. Thomas, Can E. Senkal, Philip H. Howe, Besim Ogretmen*

    *Corresponding author. Email: ogretmen{at}musc.edu

    This PDF file includes:

    • Fig. S1. CerS4 is down-regulated in most advanced metastatic tumor tissues.
    • Fig. S2. CerS4 knockdown results in increased cell migration and invasion.
    • Fig. S3. CerS4−/− mice exhibit irreversible alopecia and decreased C18- and/or C20-ceramide synthesis.
    • Fig. S4. CerS4 induction increases C18- and C20-ceramide synthesis.
    • Fig. S5. Analysis of Smad7 expression and the subcellular localization of TβRI/II after CerS4 knockdown.
    • Fig. S6. CerS4 knockdown induces Shh abundance without affecting p-Smad3 abundance.
    • Fig. S7. CerS4 knockdown enables cell migration through cross-talk between TβRI/II and Shh/Smo signaling.
    • Fig. S8. Inhibition of primary cilia formation prevents CerS4 knockdown-induced cell migration and metastasis to the liver and lung.

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    Citation: S. Gencer, N. Oleinik, J. Kim, S. Panneer Selvam, R. De Palma, M. Dany, R. Nganga, R. J. Thomas, C. E. Senkal, P. H. Howe, B. Ogretmen, TGF-β receptor I/II trafficking and signaling at primary cilia are inhibited by ceramide to attenuate cell migration and tumor metastasis. Sci. Signal. 10, eaam7464 (2017).

    © 2017 American Association for the Advancement of Science

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