Research ArticleCancer

KIF22 coordinates CAR and EGFR dynamics to promote cancer cell proliferation

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Science Signaling  30 Jan 2018:
Vol. 11, Issue 515, eaaq1060
DOI: 10.1126/scisignal.aaq1060
  • Fig. 1 CAR regulates lung cancer cell proliferation in vitro and in vivo.

    (A) Western blots of coxsackievirus and adenovirus receptor (CAR) knockdown (KD) in A549 cells, either untransfected [wild type (WT)] or transfected with control short hairpin RNA (shRNA) (−ve) or one of two shRNA sequences targeting CAR (shA and shB). (B) A549 cell proliferation over 48 hours in response to serum (left) or epidermal growth factor (EGF) (10 ng/ml; right). Data are normalized to serum-free control samples for both and are from three independent experiments. (C) Representative images (left) and analysis (right) of agar colony growth assays in A549 control or CAR KD cells (shA and shB). Scale bar, 100 μm. Data are the average number of colonies per field in all cells from 10 fields per cell line and are representative of three independent experiments. (D) Representative images of resected tumors from xenograft models using H1975 control or CAR KD cells. Graphs on the right show tumor volume and weight over time in nine (control), five (siCAR-1), or five (siCAR-2) mouse models pooled from two independent experiments. (E) Example images of phospho-histone H3 (p-histone H3; top) and Ki-67 (bottom) staining in xenograft tissues from H1975 cell tumors in (D). Scale bar, 50 μm. (F) Analysis of the p-histone H3 and Ki-67 staining in xenografts represented in (E). n ≥ 12 tumors per condition over two independent experiments. Data in all graphs are means ± SEM. *P < 0.01, **P < 0.005, ***P < 0.001 by two-way analysis of variance (ANOVA).

  • Fig. 2 CAR promotes postmitotic daughter cell attachment and spreading.

    (A) Representative images from time-lapse movies of shControl or CAR KD (shA) A549 cells undergoing division. Green arrows denote dividing cells, and magenta arrows denote initiation of daughter cell separation after division. (B) Quantification of time-lapse movies of control and CAR KD cells (shA and shB) represented in (A), assessing the time taken for cells to respread after division (left) and the percentage of cells that separate completely after division (right). Data are quantified from at least 20 cells over three independent experiments. Data are means ± SEM. *P < 0.05, ***P < 0.005. (C) Representative images from time-lapse movies of A549 cells expressing CAR–green fluorescent protein (GFP) and H2BK-mCherry; n = 4 experiments. Green arrows denote sites of high CAR-GFP at cell-cell contact points. Scale bars, 10 μm.

  • Fig. 3 CAR is phosphorylated in response to EGF, leading to CAR movement within cell junctions.

    (A) Representative images from time-lapse movies of A549 cells expressing CAR-GFP at time 0 (untreated; top) versus 30 min after EGF addition (10 ng/ml; bottom). Graph shows quantification of CAR intensity at junctions measured at 0- and 30-min time points (example analysis areas shown in zoomed regions in images are shown on the left). Data are pooled from >20 junctions from at least three independent experiments and are means ± SEM. (B) Western blots of lysates of A549 cells treated with EGF (10 ng/ml) for the specified times and probed for the specified proteins. Graph shows quantification of phosphorylated CAR (p-CAR) abundance (normalized to total CAR levels) relative to time 0 from three independent experiments. Data are means ± SEM. *P < 0.05, ***P < 0.001. p-EGFR, phosphorylated EGF receptor; p-ERK, phosphorylated extracellular signal–regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (C) Representative confocal images of A549 cells fixed after 0 or 60 min of EGF stimulation (10 ng/ml) and stained for 4′,6-diamidino-2-phenylindole (DAPI) (blue, top), phospho-CAR (white, top), and actin (bottom). Graph shows quantification of p-CAR staining from images at 5, 15 and 60 min after EGF stimulation from 100 cells per condition. Data are means ± SEM. **P < 0.005, ***P < 0.001. (D) Western blots of control or PKCδ KD A549 cells treated with EGF for the specified time periods and probed for specified antibodies. Graph shows quantification of p-CAR levels relative to time 0 from three independent experiments. Data are means ± SEM. ***P < 0.001. RFP, red fluorescent protein; PKCδ, protein kinase C δ. (E) Representative images from time-lapse movies of A549 cells expressing WT CAR-GFP (top) or AA-CAR-GFP (bottom) at 0 and 30 min after EGF stimulation. Graphs show analysis of AA-CAR-GFP movement at junctions as in (A). Data are means ± SEM. All data were analyzed for statistical differences using two-way ANOVA.

  • Fig. 4 CAR binds to KIF22.

    (A) Representative blot of pull-downs using glutathione S-transferase (GST)–tagged cytoplasmic domain of CAR incubated with lysates from A549 cells and probed for KIF22. GST alone was used a control. Coomassie-stained equivalent gel shows the input (bottom). (B) Representative blots of A549 cells expressing GFP, WT CAR-GFP, or AA-CAR-GFP and subjected to GFP immunoprecipitation (IP). Blots were probed for KIF22 or GFP as indicated. (C) A549 cells expressing CAR-GFP were serum-starved (−) or EGF-stimulated (10 ng/ml for 15 min; +) in the presence or absence of AG1478 and subjected to GFP IP followed by probing for specified proteins. Top two panels show IP complexes, and bottom three panels show input lysates. Quantifications of KIF22 levels in IP complexes below blots are mean values from three independent experiments ± SEM. IgG, immunoglobulin G. (D) Schematic cartoon of FLAG-tagged KIF22 constructs used for binding analysis. Representative levels of KIF22-FLAG binding to GST-CAR cytoplasmic domain after pull-down from transfected human embryonic kidney (HEK) 293 cell lysates are shown (right). Coomassie-stained gel shows protein input (bottom). Quantifications of KIF22 binding are provided beneath and represent mean values from four independent experiments ± SEM. (E) Schematic cartoon of GST-tagged N-terminal (NT) and C-terminal (CT) KIF22 constructs used for pull-downs. Blots show representative results from pull-downs of KIF22-FLAG–expressing HEK293T cell lysates using GST-NT-KIF22 or GST-CT-KIF22 (bottom). Data are representative of five independent experiments. (F) Representative blots from GST-tagged KIF22 CT pull-downs from cell lysates expressing KIF22-FLAG in the presence or absence of coexpressed KIF22-CCD-HALO. GST was used as a control. Coomassie-stained gel shows protein input (bottom). Values beneath show levels of KIF22-FLAG in pull-downs and represent mean values from three independent experiments ± SEM. All data were analyzed for statistical differences using two-way ANOVA.

  • Fig. 5 KIF22 promotes EGFR retention at the plasma membrane and signaling in response to EGF.

    (A) Analysis of the proliferation of A549 cells transfected with control or one of two KIF22-targeted small interfering RNAs (siRNAs) and treated with EGF and quantified relative to serum-starved control cells. Data are means ± SEM. **P < 0.005. (B) Representative blots of lysates from control or KIF22 KD A549 cells treated with EGF over the specified time periods and probed with the specified antibodies. (C) Representative blots of surface biotinylation experiments showing the amount of surface EGFR [basal unstripped (BU)] and internalized EGFR after 0, 15, and 60 min of EGF stimulation (10 ng/ml) in control or KIF22 KD A549 cells. Blots were probed with specified antibodies. Data in the graph (right) were normalized to basal unstrapped (black bars) and are means ± SEM from four independent experiments. *P < 0.01 versus equivalent time point in Ctrl samples. (D) Representative images of control or KIF22 KD A549 cells, either untreated or after stimulation with EGF (10 ng/ml for 15 min), fixed and stained for DAPI (blue) and EGFR (white). Scale bars, 10 μm. Graph shows quantification of junctional EGFR levels after stimulation with EGF (right). Data are means ± SEM from 30 cells across three independent experiments. *P < 0.01, ***P < 0.001. All data were analyzed for statistical differences using two-way ANOVA.

  • Fig. 6 KIF22 regulates EGF-dependent movement of EGFR and CAR within junctions.

    (A) Representative images of CAR-GFP from time-lapse movies of control or KIF22 KD A549 cells expressing CAR-GFP, either untreated or after 30 min of EGF stimulation. CAR-GFP movement into junctions was quantified as described in Fig. 3 (A and E) and is presented as means ± SEM. (B) Representative images of stills taken at specified time points after EGF treatment from time-lapse confocal movies of A549 cells coexpressing CAR-GFP (top), EGFR-mCherry (bottom), and control or KIF22 siRNA. (C) Quantification of movies as in (B) showing the intensity of CAR-GFP (green) and EGFR-mCherry (magenta) at junctions over time after stimulation with EGF. Data are means ± SEM from 60 cells across three independent experiments. All data were analyzed for statistical differences using two-way ANOVA. au, arbitrary units.

  • Fig. 7 KIF22 regulates microtubule organization and dynamics in cells in interphase.

    (A) Representative examples of confocal images of control or KIF22 KD A549 cells, fixed and stained for DAPI (nucleus; blue), β-tubulin (red), or F-actin (phalloidin; green). Graphs show the number of nuclei per field and the area of cells with no tubulin staining present represented as area (in square micrometers per field). Data are means ± SEM pooled from nine fields per condition across three independent experiments. *P < 0.01, ***P < 0.001. (B) Representative example images from time-lapse movies of GFP-tubulin expressed in control or KIF22 KD A549 cells after stimulation with EGF. Time-dependent changes are depicted as color scales from time-projected stacks in which blue represents regions of highly dynamic microtubule (MT) growth and white denotes regions of static or disassembling MTs. Scale bar, 2 μm. (C) Analysis of MT growth rate (in square micrometers per minute), time spent in growth phase (in seconds), and catastrophe events per minute in control or KIF22 KD cells, with and without EGF (10 ng/ml). Data are means ± SEM pooled from 22 cells in total across two independent experiments. *P < 0.05, **P < 0.005, ***P < 0.001. (D) Representative Western blots of acetylated tubulin in control and KIF22 KD cells. Data are means ± SEM from four independent experiments. **P < 0.005. All data were analyzed for statistical differences using two-way ANOVA.

  • Fig. 8 Cytoplasmic KIF22 sustains EGFR at the plasma membrane.

    (A) Western blots (top) assessing the efficiency of KIF22 KD and reexpression and representative images (bottom) of control (siCtrl) or KIF22 siRNA–treated A549 cells coexpressing FLAG-tagged D3 (N terminus) or D5 (C terminus) truncations of KIF22 and treated with EGF (10 ng/ml) for 15 min, followed by fixation and staining for DAPI (blue), FLAG (green), and EGFR (red). EGFR staining is shown as single-channel images (white) below the merged images; red stars denote FLAG-expressing cells. Scale bar, 20 μm. (B) Quantification of junctional EGFR levels after stimulation with EGF from experiments shown in (A). Data are means ± SEM pooled from at least 40 fields of view across two independent experiments. **P < 0.005. (C) Representative images of KIF22-D3 expressed in KIF22 siRNA–treated A549 cells treated with EGF for 15 min followed by fixation and staining for EGFR (magenta) and KIF22-D3-FLAG (green). White arrows denote areas of colocalized EGFR and KIF22-D3 at the plasma membrane. Scale bar, 10 μm. (D) Representative images of p-EGFR staining in KIF22 siRNA–treated cells reexpressing KIF22-D3-FLAG after EGF treatment (10 ng/ml for 15 min). Cells were fixed and stained for p-EGFR (magenta) and FLAG (green). (E) Representative images of control or KIF22 siRNA–treated CAR KD A549 cells coexpressing FLAG-tagged D3 mutants of KIF22 treated with EGF (10 ng/ml) for 15 min followed by fixation and staining for DAPI (blue), FLAG (green), and EGFR (red). Stars denote channels as described in (A). Graph shows quantification of junctional EGFR abundance after stimulation with EGF. Data are means ± SEM. **P < 0.005. All data were analyzed for statistical differences using two-way ANOVA. Scale bar, 20 μm. (F) Proposed model of KIF22 function on EGFR and CAR upon EGF binding: EGF binding to EGFR drives mitogen-activated protein kinase (MAPK) activation, PKCδ activation, and phosphorylation of CAR. EGFR activation also promotes KIF22-CAR binding and decreases KIF22-dependent microtubule dynamics. Resulting stabilized microtubules promote EGFR retention at the plasma membrane and enhance EGFR signaling.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/515/eaaq1060/DC1

    Fig. S1. CAR regulates proliferation in H1975 cells.

    Fig. S2. CAR does not regulate EGFR signaling or degradation in response to EGF.

    Fig. S3. KIF22 binds to CAR.

    Fig. S4. KIF22 regulates cell proliferation.

    Fig. S5. Blocking endocytosis does not alter A549 cell EGFR signaling.

  • Supplementary Materials for:

    KIF22 coordinates CAR and EGFR dynamics to promote cancer cell proliferation

    Rosemary Pike, Elena Ortiz-Zapater, Brooke Lumicisi, George Santis, Maddy Parsons*

    *Corresponding author. Email: maddy.parsons{at}kcl.ac.uk

    This PDF file includes:

    • Fig. S1. CAR regulates proliferation in H1975 cells.
    • Fig. S2. CAR does not regulate EGFR signaling or degradation in response to EGF.
    • Fig. S3. KIF22 binds to CAR.
    • Fig. S4. KIF22 regulates cell proliferation.
    • Fig. S5. Blocking endocytosis does not alter A549 cell EGFR signaling.

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    Citation: R. Pike, E. Ortiz-Zapater, B. Lumicisi, G. Santis, M. Parsons, KIF22 coordinates CAR and EGFR dynamics to promote cancer cell proliferation. Sci. Signal. 11, eaaq1060 (2018).

    © 2018 American Association for the Advancement of Science

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