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. 2014 Mar;8(2):207-20.
doi: 10.1016/j.molonc.2013.11.002. Epub 2013 Nov 18.

Pharmacologic inhibition of vacuolar H+ ATPase reduces physiologic and oncogenic Notch signaling

Francis Kobia  1 , Serena Duchi  1 , Gianluca Deflorian  1 , Thomas Vaccari  2
Affiliations

Pharmacologic inhibition of vacuolar H+ ATPase reduces physiologic and oncogenic Notch signaling

Francis Kobia et al. Mol Oncol. 2014 Mar.

Abstract

Notch signaling in prominently involved in growth regulation in metazoan tissues. Because of this, Notch is often upregulated in cancer and current efforts point to developing drugs that block its activation. Notch receptor endocytosis towards acidic compartments is a recently appreciated determinant of signaling activation. Vacuolar H(+) ATPase (V-ATPase) is responsible for acidification of endocytic organelles and mutants in V-ATPase subunit encoding genes in model organisms have been recently shown to display loss of Notch signaling. Here, we show that administration of BafilomycinA1 (BafA1), a highly specific V-ATPase inhibitor decreases Notch signaling during Drosophila and Zebrafish development, and in human cells in culture. In normal breast cells, we find that BafA1 treatment leads to accumulation of Notch in the endo-lysosomal system, and reduces its processing and signaling activity. In Notch-addicted breast cancer cells, BafA1 treatment reduces growth in cells expressing membrane tethered forms of Notch, while sparing cells expressing cytoplasmic forms. In contrast, we find that V-ATPase inhibition reduces growth of leukemia cells, without affecting Notch activatory cleavage. However, consistent with the emerging roles of V-ATPase in controlling multiple signaling pathways, in these cells Akt activation is reduced, as it is also the case in BafA1-treated breast cancer cells. Our data support V-ATPase inhibition as a novel therapeutic approach to counteract tumor growth via signaling pathways regulated at the endo-lysosomal level.

Keywords: BafilomycinA1; Cancer; Endocytosis; Notch; V-ATPase.

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Figures

Figure 1
Figure 1
Reduced Notch signaling in Drosophila imaginal disc upon pharmacological inhibition of V‐ATPase. (A–C) Single confocal sections of 3rd instar wing imaginal discs from larvae expressing the Notch signaling reporter E(spl)mβ‐LacZ that have been fed with the indicated drug. Discs are stained with anti‐βGal to detect expression of the Notch target. Compared with discs from mock‐fed animals (A), discs from animals fed with DAPT (B) and BafA1 (C) show a significant decrease of Beta‐Gal expression. (D) Quantitative RT‐PCR on mRNA extracted from 3rd instar wing imaginal discs from flies fed with drugs as indicated. E(spl)mβ‐LacZ and E(spl)m7 show a 30–40% decrease upon γ‐secretase inhibition and more than 60% with V‐ATPase inhibitor BafA1. (E–F) Single confocal section of discs not expressing (E) or expressing (F) Shrub:GFP under the control of RnGAL4, a wing pouch specific driver. (G–L) High magnification confocal sections of wing pouch cells not expressing (G, I, K) or expressing (H, J, L) Shrub:GFP. Discs have been stained to detect Ubiquitin (G–H), the endosomal marker Avl (I–J) or the Notch intracellular domain (NICD; K‐L). In discs expressing Shrub:GFP, accumulation of ubiquitinated cargoes, including Notch, at endosomal sites is observed. Single channels for Ubiquitin, Avl and NICD are shown in H′, J′, L′. (M) Quantitative RT‐PCR on mRNA extracted from wing imaginal discs from Shrub:GFP expressing flies fed with drugs and induced as indicated. E(spl)mβ expression is 30% reduced upon feeding with BafA1 and 50% upon γ‐secretase inhibition. Comparable GFP expression levels indicate equal amounts of Shrub:GFP expressing cells in induced samples under different drug treatment.
Figure 2
Figure 2
Impaired Notch‐signaling activity by V‐ATPase inhibition during vertebrate development. (A–E) Representative 28 hpf zebrafish embryos treated as at gastrulation as indicated and visualized in bright field, lateral view. Compared to mock‐treated embryo (A) embryos incubated with varying doses of DAPT (B–C) or BafA1 (D–E) or both (F) show mild morphological defects. (A′–F′) GFP levels associated to the expression of the Notch reporter line Tg(Tp1bglob:eGFP)ˆum14 visualized in the same embryos. Incubation with DAPT or BafA1 results in dose‐dependent reduction of GFP signal (B′–F′). Note that combination of both drugs at low dose leads to a reduction of GFP expression that is comparable to treatment with either of the drugs at high dose (compare B′ and D′ with F′).
Figure 3
Figure 3
V‐ATPase inhibition reduces Notch signaling activation in human breast cells. (A–D) Single confocal section showing the subcellular localization of endogenous Notch1 in MCF10A upon EGTA stimulation in cells treated as indicated. Cells have been stained with an anti‐Notch1, Phalloidin and DAPI were used to highlight the cell cortices and nuclei. Nuclear Notch1 localization is completely inhibited by DAPT treatment and is significantly reduced by BafA1 treatment. Single channels for anti‐Notch1 are shown in A′–D′. Nuclear pixel intensity quantification of panel A′–D′ is shown in E. (F) Western blot analysis of full length Notch1 (300 KDa) and cleaved Notch1 (cNICD1) on extracts from MCF10A treated as indicated. Stimulation with EGTA leads to production of the γ‐secretase‐cleaved active form of Notch (cNICD1), which is reduced upon pretreatment with BafA1 and abolished upon pretreatment with DAPT. (G) Quantitative RT‐PCR to detect Hes1 expression levels in MCF10A cells. Cells were pretreated with DMSO, DAPT, or BafA1 and stimulated with EGTA. Upon BafA1 pretreatment we observe a 20% reduction of Hes1 expression.
Figure 4
Figure 4
V‐ATPase inhibition impairs Notch signaling and associated growth in breast cancer cells. (A–D) Cell culture growth after 7 days upon treatment as indicated. Growth of normal breast MCF10A cells and breast cancer HCC1187 cells, which harbor a translocation leading to expression of a cytoplasmic active Notch truncation, is not affected by drug treatments. In contrast, growth of breast cancer HCC2218 and HCC1599 cells, which harbor translocations leading to expression of a membrane tethered, active Notch truncation, is sensitive to both DAPT and BafA1 in a dose‐sensitive fashion. Note that combination of both drugs at low dose in HCC2218 cells results in a growth inhibition that is comparable to treatment with either of the drugs at high dose. (E–F) Western blot analysis of cleaved Notch1 (cNICD1) on extracts from HCC1599 and HCC2218 treated for 7 days as indicated. cNICD1 production is strongly reduced upon pretreatment with BafA1 and DAPT. (G) Quantitative RT‐PCR to detect Hes1 expression levels in HCC1599 cells treated as indicated. Upon BafA1 pretreatment we observe a 25% reduction of Hes1 expression.
Figure 5
Figure 5
V‐ATPase inhibition reduces growth of T‐cell leukemia cell lines. (A) Cell culture growth after 7 days upon treatment as indicated. Growth of T‐cell leukemia DND‐41 cells, which harbor activating Notch1 mutations, is sensitive to both DAPT and BafA1 in a dose‐sensitive fashion. Note that combination of both drugs at low dose results in a growth inhibition that is comparable to treatment with either of the drugs at high dose. (B–C) Western blot analysis of cNICD1 (B) or of Akt and pAKT S473 (C) on extracts from DND‐41 cells treated as indicated. cNICD1 production is not reduced upon pretreatment with BafA1. In contrast, pAKT S473 levels are reduced upon pretreatment with BafA1 and DAPT. (D–E) Cell culture growth after 7 days upon treatment as indicated. Growth of T‐cell leukemia DND‐41 cells (D) and of CCRF‐CEM cells (E), which also harbor inactivating PTEN mutations, is sensitive to the Akt signaling inhibitor SH6, but not to GSIs. Note that combination of SH6 with BafA1 results in maximal growth inhibition. (F–G) Western blot analysis of cNICD1 (F) or of Akt and pAKT S473 (G) on extracts from CCRF‐CEM cells treated as indicated. cNICD1 production and pAKT levels are not reduced upon pretreatment with BafA1.
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References

    1. Agrawal, N. , Frederick, M.J. , Pickering, C.R. , Bettegowda, C. , Chang, K. , Li, R.J. , Fakhry, C. , Xie, T.-X. , Zhang, J. , Wang, J. , Zhang, N. , El-Naggar, A.K. , Jasser, S.A. , Weinstein, J.N. , Treviño, L. , Drummond, J.A. , Muzny, D.M. , Wu, Y. , Wood, L.D. , Hruban, R.H. , Westra, W.H. , Koch, W.M. , Califano, J.A. , Gibbs, R.A. , Sidransky, D. , Vogelstein, B. , Velculescu, V.E. , Papadopoulos, N. , Wheeler, D.A. , Kinzler, K.W. , Myers, J.N. , 2011. Exome sequencing of head and neck squamous cell carcinoma reveals inactivating mutations in NOTCH1. Science. 333, 1154–1157. - PMC - PubMed
    1. Alessi, D.R. , Andjelkovic, M. , Caudwell, B. , Cron, P. , Morrice, N. , Cohen, P. , Hemmings, B.A. , 1996. Mechanism of activation of protein kinase B by insulin and IGF-1. Embo J.. 15, 6541–6551. - PMC - PubMed
    1. Amsterdam, A. , Nissen, R.M. , Sun, Z. , Swindell, E.C. , Farrington, S. , Hopkins, N. , 2004. Identification of 315 genes essential for early zebrafish development. Proc. Natl. Acad. Sci. U. S. A.. 101, 12792–12797. - PMC - PubMed
    1. Aste-Amezaga, M. , Zhang, N. , Lineberger, J.E. , Arnold, B.A. , Toner, T.J. , Gu, M. , Huang, L. , Vitelli, S. , Vo, K.T. , Haytko, P. , Zhao, J.Z. , Baleydier, F. , L'Heureux, S. , Wang, H. , Gordon, W.R. , Thoryk, E. , Andrawes, M.B. , Tiyanont, K. , Stegmaier, K. , Roti, G. , Ross, K.N. , Franlin, L.L. , Wang, F. , Chastain, M. , Bett, A.J. , Audoly, L.P. , Aster, J.C. , Blacklow, S.C. , Huber, H.E. , 2010. Characterization of Notch1 antibodies that inhibit signaling of both normal and mutated Notch1 receptors. PLoS One. 5, e9094 - PMC - PubMed
    1. Ayyanan, A. , Civenni, G. , Ciarloni, L. , Morel, C. , Mueller, N. , Lefort, K. , Mandinova, A. , Raffoul, W. , Fiche, M. , Dotto, G.P. , Brisken, C. , 2006. Increased Wnt signaling triggers oncogenic conversion of human breast epithelial cells by a Notch-dependent mechanism. Proc. Natl. Acad. Sci. U. S. A.. 103, 3799–3804. - PMC - PubMed

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