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UDP-glucose ceramide glucosyltransferase activates AKT, promoted proliferation, and doxorubicin resistance in breast cancer cells

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Abstract

The UDP-glucose ceramide glucosyltransferase (UGCG) is a key enzyme in the synthesis of glycosylated sphingolipids, since this enzyme generates the precursor for all complex glycosphingolipids (GSL), the GlcCer. The UGCG has been associated with several cancer-related processes such as maintaining cancer stem cell properties or multidrug resistance induction. The precise mechanisms underlying these processes are unknown. Here, we investigated the molecular mechanisms occurring after UGCG overexpression in breast cancer cells. We observed alterations of several cellular properties such as morphological changes, which enhanced proliferation and doxorubicin resistance in UGCG overexpressing MCF-7 cells. These cellular effects seem to be mediated by an altered composition of glycosphingolipid-enriched microdomains (GEMs), especially an accumulation of globotriaosylceramide (Gb3) and glucosylceramide (GlcCer), which leads to an activation of Akt and ERK1/2. The induction of the Akt and ERK1/2 signaling pathway results in an increased gene expression of multidrug resistance protein 1 (MDR1) and anti-apoptotic genes and a decrease of pro-apoptotic gene expression. Inhibition of the protein kinase C (PKC) and phosphoinositide 3 kinase (PI3K) reduced MDR1 gene expression. This study discloses how changes in UGCG expression impact several cellular signaling pathways in breast cancer cells resulting in enhanced proliferation and multidrug resistance.

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Acknowledgements

This work was funded by the Deutsche Forschungsgemeinschaft (WE 5825/1-1), SFB 1039 TP B05, the August Scheidel-Stiftung, the Heinrich und Fritz Riese-Stiftung, and Minerva-Stiftung. The supports by the Ministerium für Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen, the Senatsverwaltung für Wirtschaft, Technologie und Forschung des Landes Berlin, and the Bundesministerium für Bildung und Forschung and BMBF (Code 031L0108A, 031A534B) are also gratefully acknowledged. In addition, funding by the Sigrid Jusélius Foundation and Magnus Ehrnrooth Foundation and Åbo Akademi University are acknowledged.

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Authors and Affiliations

  1. pharmazentrum frankfurt/ZAFES, Institute of Clinical Pharmacology, Johann Wolfgang Goethe University, House 74, Theodor Stern-Kai 7, 60590, Frankfurt am Main, Germany

    Marthe-Susanna Wegner, Nina Schömel, Lisa Gruber, Stephanie Beatrice Örtel, Sandra Trautmann, Gerd Geisslinger & Sabine Grösch

  2. Biochemistry, Faculty of Science and Engineering, Åbo Akademi University, Artillerigatan 6A, III, BioCity, 20520, Turku, Finland

    Matti Aleksi Kjellberg & Peter Mattjus

  3. Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Project Group Translational Medicine and Pharmacology (TMP), Frankfurt am Main, Germany

    Jennifer Kurz & Gerd Geisslinger

  4. Leibniz-Institut für Analytische Wissenschaften, ISAS e. V., Otto-Hahn-Straße 6b, 44227, Dortmund, Germany

    Bing Peng & Robert Ahrends

  5. Institute of Biochemistry II, Johann Wolfgang Goethe University, Theodor Stern-Kai 7, 60590, Frankfurt am Main, Germany

    Martin Wegner & Manuel Kaulich

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  1. Marthe-Susanna Wegner
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Correspondence to Marthe-Susanna Wegner.

Electronic supplementary material

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18_2018_2799_MOESM1_ESM.pptx

Supplementary material 1 (PPTX 553 kb) Supplemental 1 Stable UGCG knockdown in MCF-7 cells by CRISPR/Cas. (A) Validation of the UGCG knockdown by qRT-PCR, Western blot analysis and immunocytochemistry. Data are represented as a mean of n = 4 to 11 ± SEM. Unpaired t test with Welch’s correction. (B) Transmitted light image acquisition show a reduced cytoplasm size in MCF-7/UGCG KD cells as compared to control and MCF-7/UGCG OE cells. Representative images. (C) Determination of the relative living cell number of MCF-7 cells. Data are represented as a mean of n = 7 to 15 ± SEM. Unpaired t test with Welch’s correction. p ≤ * 0.05, p ≤ ** 0.01, p ≤ *** 0.001, p ≤ **** 0.0001

18_2018_2799_MOESM2_ESM.pptx

Supplementary material 2 (PPTX 489 kb) Supplemental 2 Cytoskeleton, cytoplasm staining and MDR1 mRNA expression of MCF-7 cells. (A) Immunocytochemistry of MCF-7 cells. Cytoskeleton is indicated as β-actin staining (upper panel). Cell membrane is indicated as β-catenin staining (lower panel). Nuclei are stained by DAPI. (B) Analysis of MDR1 mRNA expression by qRT-PCR. The expression is related to the housekeeping gene RPL37A. Data are represented as a mean of n = 3-6 ± SEM. Unpaired t test with Welch’s correction. p ≤ *** 0.001

18_2018_2799_MOESM3_ESM.pptx

Supplementary material 3 (PPTX 487 kb) Supplemental 3 Analysis of mRNA expression of sphingolipid enzymes by qRT-PCR and sphingolipid concentration determination by LC–MS/MS in 3D MCF-7 spheroids. (A) Analysis of sphingolipid enzymes mRNA expression by qRT-PCR. The expression is related to the housekeeping gene RPL37A. Data are represented as a mean of n = 3 ± SEM. (B) Concentrations of C14:0-, C16:0-, C18:0-, C18:1-, C20:0-, C24:0-Cer determined by LC–MS/MS. (C) Concentrations of C16:0-, C18:0-, C18:1-, C24:1-GlcCer, C16:0-, C18:0-, C24:0- and C24:1-LacCer determined by LC–MS/MS. (D) Total Cer, Glc- and LacCer levels determined by LC–MS/MS. Unpaired t test with Welch’s correction. p ≤ * 0.05, p ≤ ** 0.01, p ≤ *** 0.001, p ≤ **** 0.0001

18_2018_2799_MOESM4_ESM.pptx

Supplementary material 4 (PPTX 16050 kb) Supplemental 4 Analysis of the size and nuclei staining of 3D MCF-7 spheroids by microscopy. The transmitted light microscopy shows that MCF-7/UGCG OE spheroids are more densely packed than MCF-7/naiv and MCF-7/pTarget cells. DAPI staining indicates cell nuclei. Representative images

18_2018_2799_MOESM5_ESM.pptx

Supplementary material 5 (PPTX 283 kb) Supplemental 5 Determination of sphingolipid concentrations in MCF-7 cells by LC–MS/MS. (A) Sph-1p concentrations in MCF-7/pTarget and MCF-7/UGCG OE cells following 0 and 2 µM PPMP stimulation. (B) GEMs were isolated and total sphinganine, Cer and LacCer levels of fractions 1 to 10 were determined by LC–MS/MS. Data are represented as a mean of n = 3 ± SEM. p ≤ * 0.05

18_2018_2799_MOESM6_ESM.pptx

Supplementary material 6 (PPTX 442 kb) Supplemental 6 Analysis of different cellular marker in fractions 1 to 10 in MCF-7 cells by Western blot analysis. (A) PonceauS staining. (B) Western blot analysis of different cellular marker

18_2018_2799_MOESM7_ESM.pptx

Supplementary material 7 (PPTX 427 kb) Supplemental 7 Analysis of phosphorylation status of key signaling molecules in MCF-7 cells following PPMP stimulation by an antibody-based array. Array was performed according to manufacturer’s protocol. (A) Densitometrical analysis of the array. Data are represented as a mean of n = 2 ± SEM. RFU = relative fluorescence unit. (B) Representative arrays were displayed. p ≤ * 0.05, p ≤ ** 0.01, p ≤ **** 0.0001

18_2018_2799_MOESM8_ESM.pptx

Supplementary material 8 (PPTX 427 kb) Supplemental 8 Analysis of phosphorylation status of key signaling molecules in 3D MCF-7 spheroids by an antibody-based array. Array was performed according to manufacturer’s protocol. (A) Densitometrical analysis of the array. Data are represented as a mean of n = 4 ± SEM. (B) Representative arrays were displayed. RFU = relative fluorescence unit. RFU = relative fluorescence unit. p ≤ * 0.05, p ≤ **** 0.0001

18_2018_2799_MOESM9_ESM.pptx

Supplementary material 9 (PPTX 588 kb) Supplemental 9 Analysis of phosphorylation status of key signaling molecules in MCF-7/UGCG KD cells by an antibody-based array. Array was performed according to manufacturer’s protocol. (A) Densitometrical analysis of the array. Data are represented as a mean of n = 5-7 ± SEM. RFU = relative fluorescence unit. (B) Representative arrays were displayed. p ≤ * 0.05

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Wegner, MS., Schömel, N., Gruber, L. et al. UDP-glucose ceramide glucosyltransferase activates AKT, promoted proliferation, and doxorubicin resistance in breast cancer cells. Cell. Mol. Life Sci. 75, 3393–3410 (2018). https://doi.org/10.1007/s00018-018-2799-7

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