Centrosome-declustering drugs mediate a two-pronged attack on interphase and mitosis in supercentrosomal cancer cells

Abstract

Classical anti-mitotic drugs have failed to translate their preclinical efficacy into clinical response in human trials. Their clinical failure has challenged the notion that tumor cells divide frequently at rates comparable to those of cancer cells in vitro and in xenograft models. Given the preponderance of interphase cells in clinical tumors, we asked whether targeting amplified centrosomes, which cancer cells carefully preserve in a tightly clustered conformation throughout interphase, presents a superior chemotherapeutic strategy that sabotages interphase-specific cellular activities, such as migration. Herein we have utilized supercentrosomal N1E-115 murine neuroblastoma cells as a test-bed to study interphase centrosome declustering induced by putative declustering agents, such as Reduced-9-bromonoscapine (RedBr-Nos), Griseofulvin and PJ-34. We found tight 'supercentrosomal' clusters in the interphase and mitosis of ~80% of patients' tumor cells with excess centrosomes. RedBr-Nos was the strongest declustering agent with a declustering index of 0.36 and completely dispersed interphase centrosome clusters in N1E-115 cells. Interphase centrosome declustering caused inhibition of neurite formation, impairment of cell polarization and Golgi organization, disrupted cellular protrusions and focal adhesion contacts-factors that are crucial prerequisites for directional migration. Thus our data illustrate an interphase-specific potential anti-migratory role of centrosome-declustering agents in addition to their previously acknowledged ability to induce spindle multipolarity and mitotic catastrophe. Centrosome-declustering agents counter centrosome clustering to inhibit directional cell migration in interphase cells and set up multipolar mitotic catastrophe, suggesting that disbanding the nuclear-centrosome-Golgi axis is a potential anti-metastasis strategy.

Figure 1

Clinical tumors show rampant centrosome amplification and clustering in interphase cells. (a) Representative immunofluorescence confocal micrographs showing centrosome amplification and clustering status in normal adjacent (left panel) and tumor tissues (right panel) from 10 patients of each cancer type. Insets show clustered centrosomes in representative mitotic cells (top inset) and interphase (bottom inset) in tumor samples and normal centrosomes in the normal samples. White arrows depict centrosome clusters in interphase cells. Centrosomes and microtubules were visualized by immunostaining for γ-tubulin (green) and α-tubulin (red), respectively. DNA was 4,6-diamidino-2-phenylindole (DAPI) stained (blue). (bi and bii) Quantitative bar graphs representing the percentage of centrosome amplification and the percentage of interphase cells with amplified centrosomes that exhibit centrosome clustering, respectively, in the corresponding patient tissue samples. Centrosomes were counted in interphase cells from randomly selected fields totaling at least 200 cells per sample. (c) Representative immunofluorescence confocal micrographs showing centrosome amplification and clustering status during interphase in MCF-10A, MDA-MB-231, PC-3 and HT-29 cell lines. Insets show amplified and clustered/declustered centrosomes in interphase cells. Centrosomes and microtubules were visualized by immunostaining for γ-tubulin (green) and α-tubulin (red), respectively. DNA was DAPI stained (blue). (di and dii) Quantitative bar graphs representing the percentage of centrosome amplification and the percentage of cells with amplified centrosomes that exhibit centrosome clustering, respectively, in the corresponding cell lines. Centrosomes were counted in interphase cells from randomly selected fields totaling at least 200 cells per cell line. P<0.05. Scale bar, 5 μm. BC=breast cancer, PC=prostate cancer, COL=colon cancer

Figure 2

Cell cycle phase characterization of N1E-115 cells. (a) Representative immunofluorescence confocal micrographs depicting centrosome status in all cell cycle phases of N1E-115 cells. N1E-115 cells in interphase possess an enormous number of centrosomes as evident by γ-tubulin immunostaining (green). We acquired images as z-stacks with the slice interval of 0.40 μm. Z-stack slices encompassing the entire depth of the cell were then merged, and γ-tubulin-positive spots were counted in interphase cells from randomly selected fields totaling 200 interphase cells. (b) Representative immunofluorescence confocal micrographs showing lamin A/C staining (red) across all cell cycle phases to visualize nuclear membrane in order to distinguish interphase declustering from prophase scattering of centrosome cluster. (c) Confocal micrograph of a metaphase cell stained with CREST antibodies (white), antibodies against α-tubulin (red) and γ-tubulin (green) and 4,6-diamidino-2-phenylindole (DAPI; blue) to detect microtubule-kinetochore attachments and DNA in a pseudobipolar mitotic spindle. (d) Representative micrograph of an anaphase cell immunostained for α-tubulin (red), γ-tubulin (green) and DAPI (blue) showing lagging chromosomes. Scale bar, 5 μm

Figure 3

Interphase declustering induced by centrosome-declustering agents. (a) Confocal micrographs showing interphase declustering induced by 6-h treatment with RedBr-Nos (10 μM), Griseofulvin (50 μM), PJ-34 (25 μM) and Paclitaxel (0.1 μM). The percentages indicate proportion of interphase cells with declustered centrosomes. (bi) 3-D representation and quantitative volume analysis of control and drug-treated interphase cells using the Volocity 6.3 software. Cells were co-immunostained for lamin A/C (red) and γ-tubulin (green), and z-stacks were acquired with a 0.35 μm z-step. Z-stack slices were then used to construct a 3-D image, and ROIs were defined to generate DI measurements. (bii) Quantitative bar graph representing the DI of the four drugs. P<0.05. (c) Confocal micrographs showing spindle multipolarity induced by 18h treatment with declustering agents RedBr-Nos (10 μM), Griseofulvin (50 μM), PJ-34 (25 μM) and Paclitaxel (0.1 μM). Cells were co-immunostained for α-tubulin (red) and γ-tubulin (green). The percentages indicate proportion of mitotic cells with declustered centrosomes. (d) Confocal micrographs showing Golgi dispersal concomitant with interphase declustering upon 6-h treatment with all the four declustering agents at the stated concentrations. Cells were co-immunostained for GM130 (red) and γ-tubulin (green). DNA was DAPI (4,6-diamidino-2-phenylindole) stained. Scale bar, 5 μm

Figure 4

Inhibition of neuritogenesis by centrosome-declustering agents. (a) Phase-contrast images of N1E-115 cells in SS medium showing neurite formation after 48 h of SS or with RedBr-Nos (5 μM), Griseofulvin (10 μM), PJ-34 (10 μM) and Paclitaxel (0.05 μM) treatment. Scale bar, 10 μm. (bi and bii) Quantitative bar graphs representing the average length of neurites and the percentage of population of cells showing neurite length >10 μm, respectively. Hundred cells were counted in each case. P<0.05. (c) Confocal micrographs showing neurite outgrowth after 48 h without SS, with SS or SS along with drug treatment, respectively. Cells were co-immunostained for α-tubulin (red) and γ-tubulin (green). DNA was 4,6-diamidino-2-phenylindole (DAPI) stained. (di) Immunoblot showing the CLASP1 expression levels in control and CLASP1 siRNA-transfected N1E-115 cells. (dii) Confocal micrographs showing Golgi network immunostained for GM130 (red) and centrosome cluster immunostained for γ-tubulin (green) in control and CLASP1 siRNA-transfected N1E-115 cells. (diii) Confocal micrographs showing neurite outgrowth in control and CLASP1 siRNA-transfected N1E-115 cells. (e) Confocal micrographs showing vinculin localization during neurite outgrowth after 48 h without SS, with SS or SS along with drug treatment, respectively. Cells were stained for F-actin using rhodamine-phalloidin, immunostained for vinculin (green) and DNA was DAPI stained. Scale bar, 5 μm

Figure 5

Inhibited migration induces interphase cell death or pushes cells into catastrophic mitosis. (a) Confocal micrographs showing proliferative cells with 24 h of SS or SS along with RedBr-Nos (5 μM), Griseofulvin (10 μM) and PJ-34 (10 μM) treatment, respectively. Cells were co-immunostained for Ki67 (red) and γ-tubulin (green). The percentages show proportion of Ki67-positive cells. Scale bar, 5 μm. Confocal micrographs showing cells undergoing apoptosis with 9 h of SS or SS along with RedBr-Nos (5 μM), Griseofulvin (10 μM) and PJ-34 (10 μM) treatment, respectively. Cells were co-immunostained for cleaved caspase-3 (red) and α-tubulin (green). E percentages show proportion of cells that stained positive for cleaved caspase-3. Scale bar, 10 μm. (b) Quantitative bar graphs representing the percentage of Ki67- and caspase-3-positive cells when treated with the respective drugs. Two hundred cells were counted in each case. P<0.05