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. 2021 Oct 26;12(22):2234-2251.
doi: 10.18632/oncotarget.28089.

Dishevelled-1 DIX and PDZ domain lysine residues regulate oncogenic Wnt signaling

Monica Sharma  1 , Isabel Castro-Piedras  1 , Fahmida Rasha  1 , Sabarish Ramachandran  2 , Souad R Sennoune  2 , Kathryn Furr  1 , Sharilyn Almodovar  1 , Vadivel Ganapathy  2 , Matthew B Grisham  1 , Rakhshanda Layeequr Rahman  3 , Kevin Pruitt  1
Affiliations

Dishevelled-1 DIX and PDZ domain lysine residues regulate oncogenic Wnt signaling

Monica Sharma et al. Oncotarget. .

Abstract

DVL proteins are central mediators of the Wnt pathway and relay complex input signals into different branches of the Wnt signaling network. However, molecular mechanism(s) that regulate DVL-mediated relay of Wnt signals still remains unclear. Here, for the first time, we elucidate the functional significance of three DVL-1 lysines (K/Lys) which are subject to post-translational acetylation. We demonstrate that K34 Lys residue in the DIX domain regulates subcellular localization of β-catenin, thereby influencing downstream Wnt target gene expression. Additionally, we show that K69 (DIX domain) and K285 (PDZ domain) regulate binding of DVL-1 to Wnt target gene promoters and modulate expression of Wnt target genes including CMYC, OCT4, NANOG, and CCND1, in cell line models and xenograft tumors. Finally, we report that conserved DVL-1 lysines modulate various oncogenic functions such as cell migration, proliferation, cell-cycle progression, 3D-spheroid formation and in-vivo tumor growth in breast cancer models. Collectively, these findings highlight the importance of DVL-1 domain-specific lysines which were recently shown to be acetylated and characterize their influence on Wnt signaling. These site-specific modifications may be subject to regulation by therapeutics already in clinical use (lysine deacetylase inhibitors such as Panobinostat and Vorinostat) or may possibly have prognostic utility in translational efforts that seek to modulate dysfunctional Wnt signaling.

Keywords: chromatin immunoprecipitation (ChIP); dishevelled (DVL); gene expression; lysine residue; post-translational modification.

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Conflict of interest statement

CONFLICTS OF INTEREST Authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1. High DVL-1 expression promotes cell proliferation and xenograft tumor growth in triple-negative breast cancer.
(A) Merge of immunofluorescence staining for DVL-1 (red) and nucleus (blue) in different primary human breast cancer tissues (including non-cancer adjacent tissue, luminal A, luminal B, HER2+, and basal/TNBC). (B) Graphical representation of average DVL-1 staining from multiple breast cancer patients in various subgroups (n ≥ 3; * p < 0.05; # p < 0.1). Western blot for DVL-1 protein expression in NTC and shDVL1 knockdown in (C) MDA-MB-231 and (D) MDA-MB-468 cells. Graph below represents cell proliferation rate quantified in real time by live cell imaging using the IncuCyte ZOOM (Essen Biosciences). Images were collected every 2 hours for 150 hours. (E) Western blot for DVL-1 expression in empty vector (EV) versus wild-type DVL1 gain-of-function in MDA-MB-231. (F) Cell proliferation in EV versus WT-DVL1 cells quantified in real time by live cell imaging, where images were collected every 2 hours for 80 hours. (G) Mean tumor volume of EV and WT-DVL1 MDA-MB-231 cells subcutaneously injected in mammary fat pad of immunocompromised mice (n = 5 per group, p < 0.05). Please refer to Supplementary Figure 2 for additional information for western blot panels.
Figure 2
Figure 2. K34 residue regulates DVL-1 protein-protein interaction and entry of β-catenin into the nucleus, while K69 and K285 are critical for DVL-1 subcellular localization.
(A) Western blot analysis of lysates from HEK293 cells stably expressing empty vector (EV), N-terminal HA-epitope tagged DVL-1 wild type (WT), HA-tagged deacetylation mutants (K to R), HA-tagged acetylation mutants (K to Q) on highly conserved lysine residues namely, K34, K69 and K285, probed with antibody as indicated. Also see Supplementary Figure 6 for additional information for western blot panels. (B) Immunofluorescence staining of empty vector (EV), N-HA-tagged DVL-1 (WT), K34R mutant (N-HA-K34R), K34Q mutant (N-HA-K34Q), K69R mutant (N-HA-K69R), K69Q mutant (N-HA-K69Q), K285R mutant (N-HA-K285R), K285Q mutant (N-HA-K285Q), proteins in stably expressing HEK293 cells. Merge of N-HA-DVL-1 (red) and nuclear staining (blue) proteins is shown as HA/DAPI for each of the mutant. Merge of actin (green) and N-HA-DVL-1 (red) proteins is shown as HA/Actin for each of the mutant. For co-immunoprecipitation (Co-IP) experiments, HEK293 cells stably expressing DVL-1 mutants at K34 residues were used where we immunoprecipitated using antibodies against (C) HA-epitope and blotted for binding partners such as DVL-2 and casein kinase 1 (CK1e) versus (D) a reciprocal Co-IP was performed to pull down different binding partners such as Axin1, DVL3, β-catenin, FZD7 and blotted for HA-tag. Empty vector (EV) was used as a negative control, and whole cell extracts (WCE) as a positive control. IgG heavy chain (HC) was blotted for as a control for equal antibody loading for immunoprecipitation. β-actin was included as a loading control for WCE. See Supplementary Figure 7 for additional information for western blot panels. (E) Whole (W), cytoplasmic (C) and nuclear (N) extracts from HEK293 cells stably expressing EV, WT-DVL1, K34R, and K34Q were analyzed using Western blots. The blots were probed with antibodies against active (non-phosphorylated) β-catenin, total β-catenin, and HA-tag. Lamin was used as a control for nuclear extract and β-tubulin was used as a control for cytosolic proteins. See Supplementary Figure 8 for additional information for western blot panels.
Figure 3
Figure 3. Conserved K34 residue on DVL-1 regulates canonical WNT target gene expression in various cellular models.
mRNA expression of WNT target genes such as AXIN2, NANOG, SOX17, OCT4, FZD7, and CYCLIND1 was determined by qRT-PCR (normalized to B2M as control) in (A) HEK293 and (B) MDA-MB-231 cells stably expressing EV, WT-DVL1, K34R, and K34Q mutants. The sign “*” represents significant change in gene expression between WT and mutants, while “#” represents significant change between R and Q mutants. All results are expressed as mean ± SEM and considered significant at */# p < 0.05, **/## p < 0.01 and ***/### p < 0.001. (C) Schematic representation of the functional significance of DVL1-K34 residue with respect of Wnt signaling. Mutation on highly conserved K34 residue impacts DVL-1 protein scaffolding property, entry of β-catenin into the nucleus, thereby downregulating canonical WNT target gene expression.
Figure 4
Figure 4. K69 and K285 lysine residues influences its binding and regulation of WNT target genes in triple-negative breast cancer models.
Occupancy of HA-tagged DVL-1 at promoters of c-myc and cyclinD1 gene were analysed by real-time PCR. ChIP experiments for HA-tag were performed in (A) MDA-MB-231 and (B) MDA-MB-468 cells stably expressing EV (empty vector; negative control), HA-tagged wild-type DVL-1 (WT), HA-tagged deacetylation mutants (K69R and K285R), and HA-tagged acetylation mutants (K69Q and K285Q). The sign “*” represents significant change in HA-tagged DVL-1 binding between WT and mutants. (C) mRNA expression of WNT target genes such as C-MYC, CYCLIND1, AXIN2, NANOG, SOX17, and OCT4, was determined by qRT-PCR (mean ± SEM, normalized to β-actin as control) in triple-negative breast cancer cells (MDA-MB-231) stably expressing DVL-1 K69 and K285 mutants. The sign “*” represents significant change in gene expression between WT and mutants, while “#” represents significant change between R and Q mutants. All results are expressed as mean ± SEM and considered significant at */# p < 0.05, **/## p < 0.01 and ***/### p < 0.001.
Figure 5
Figure 5. Conserved DVL-1 lysines at DIX and PDZ domain regulates cell migration and cell cycle progression in breast cancer cells.
(A) MDA-MB-231 cells expressing different DVL-1 mutants were analyzed in a transwell migration assay and images were taken using Nikon microscope. (B) Changes in migration were measured after 20h and cells were counted using AdipoGauge software. All results are expressed as mean ± SD and considered significant at * p < 0.05 and ** p < 0.01. (C) The effect of DVL-1 mutants was analyzed in a cell cycle assay using a Vybrant cell violet dye from Invitrogen followed by flow cytometry. The live cells are represented in the SSC versus FSC plots, which were further distributed among different cell cycle stages such as SubG1, G1, S, G2/M, and Super-G2 phase. Changes in cell cycle stages from four-independent experiments are represented in the table. The sign “*” represents significant change in gene expression with respect to WT-DVL1, while “#” represents significant change between R and Q mutants. All results are expressed as mean (n = 4) and considered significant at **/## p < 0.01 and ***/### p < 0.001.
Figure 6
Figure 6. K69 and K285 residues significantly influence rate of cell proliferation and xenograft tumor growth respectively in MDA-MB-231 cells.
(A) MDA MB-231 cells were seeded onto 96 well plates and grown in the presence of nuclear stain NucLight Rapid Red Reagent (Essen Biosciences). Cells were monitored and stained nuclei were quantified in real time by live cell imaging, with images captured every 2 hours for 120 hours. Images shown here represent confluency (upper panel) versus nuclei stain (bottom panel). (B) Each dot represents the nuclear counts at each time point. Data are shown as the mean nuclear counts (red fluorescent bodies/mm2) ± SEM and considered significant at *** p < 0.001 compared to WT-DVL1. Spheroid formation assay was conducted in MDA-MB-231 cell lines stably expressing DVL-1 (C) K69 and (D) K285 mutants. Quantification of the area of spheroids is presented as the mean ± SD, where n ≥ 5 showing statistical difference after 200h *** p < 0.001. (E) Mean tumor weight of WT-DVL1, K285R and K285Q MDA-MB-231 cells subcutaneously injected in mammary fat pad of immunocompromised mice (n = 4 per group, “*” represents significant change between R and Q mutants where * p < 0.05). (F) Representative images for tumors from mice receiving WT-DVL1, K285R and K285Q MDA-MB-231 cells (top to bottom). (G) mRNA expression of WNT target genes such as CMYC, CYCLIND1, and AXIN2 was determined by qRT-PCR (mean ± SEM, normalized to β-actin as control) using tumor obtained from xenograft studies, where n = 4. The sign “*” represents significant change in gene expression. All results are expressed as mean ± SEM and considered significant at * p < 0.05, ** p < 0.01 and *** p < 0.001. (H) Schematic representation of the functional significance of lysine residues on the DIX and PDZ domains od DVL-1 with respect of Wnt signaling. While highly conserved K34 residue is critical for the entry of β-catenin into nucleus, K69/K285 residue regulate DVL-1 binding to promoter binding at Wnt target genes, and repressing their expression thereby impact broad cellular functions.
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