KIF24 depletion induces clustering of supernumerary centrosomes in PDAC cells

Abstract

Clustering of supernumerary centrosomes, which potentially leads to cell survival and chromosomal instability, is frequently observed in cancers. However, the molecular mechanisms that control centrosome clustering remain largely unknown. The centrosomal kinesin KIF24 was previously shown to restrain the assembly of primary cilia in mammalian cells. Here, we revealed that KIF24 depletion suppresses multipolar spindle formation by clustering centrosomes in pancreatic ductal adenocarcinoma (PDAC) cells harboring supernumerary centrosomes. KIF24 depletion also induced hyper-proliferation and improved mitotic progression in PDAC cells. In contrast, disruption of primary cilia failed to affect the proliferation and spindle formation in KIF24-depleted cells. These results suggest a novel role for KIF24 in suppressing centrosome clustering independent of primary ciliation in centrosome-amplified PDAC cells.

Graphical abstract

Figure S1.. Mutation and expression of KIF24 in KIF24-depleted PDAC cells.

(A, B) Panc1 or CFPAC1 cells transiently transfected with siLuciferase or siKif24 were cultured in serum-starved medium for 48 h. (A) Relative amount of KIF24 mRNA was determined using quantitative PCR. GAPDH was used as a control. Average of three independent experiments is shown. (B) Cells were immunostained as described in Fig 1B. The percentage of ciliated cells was determined. The average of three to four independent experiments is shown; >250 cells were scored each time. (A, B, C, D) (upper) Four different mutations (alleles A, B, C, and D) of the KIF24 gene in Kif24-3 Panc1 cells (note that Panc1 cells are frequently multiploid). Red sequences represent the target of gRNAs with underlined protospacer adjacent motif (PAM). (Lower) Proteins translated from each clone. Green shows incorrect residues. (D, E) shKif24-expressed Panc1 cells (D) or MiaPaCa2 and Hs766t cells (E) were cultured in serum-fed medium for 48 h. Relative amount of KIF24 mRNA was determined using quantitative PCR. GAPDH was used as a control. Average of three independent experiments is shown. (B, E) Kif24-primer2 pairs were used. (A, B, D, E) All data are shown as mean ± SD. Two-tailed t test. **P < 0.01; *P < 0.05.

Figure 1.. KIF24 depletion restores primary cilia in Panc1 cells.

(A) The indicated Panc1 cells were cultured in serum-fed medium for 48 h. Cell extracts were immunoblotted with an anti-KIF24-2 antibody. β-Actin was used as a loading control. (B, C) The indicated Panc1 cells were cultured in serum-fed (FBS+) or serum-starved medium (FBS−) for 48 h and immunostained with an anti-Arl13b antibody (green). (B) Representative images of cells in FBS− cultivation. Arrows indicate primary cilia. DNA was stained with Hoechst (blue). Scale bar, 10 μm. (C) The percentage of ciliated cells was determined. The average of four to seven independent experiments is shown; >250 cells were scored each time. (D) The indicated Panc1 cells were cultured and immunoblotted as described in Fig 1A. (E) The indicated Panc1 cells were cultured and immunostained as described in Fig 1B. The percentage of ciliated cells was determined. The average of three to four independent experiments is shown; >250 cells were scored each time. (C, E) All data are shown as mean ± SD. Two-tailed t test. **P < 0.01; *P < 0.05. Source data are available for this figure.

Figure 2.. KIF24 depletion enhances proliferation of Panc1 cells in vitro.

(A, B) The indicated Panc1 cells were cultured for 72 or 144 h, and the number of surviving cells was counted with a hemocytometer. The average of six (A) or four to five (B) independent experiments is shown. (C) The indicated Panc1 cells were cultured in serum-fed medium for 48 h and immunostained with an anti-Ki67 antibody. The quantified fluorescence intensity of Ki67 in the nucleus is shown. n = 160 (Panc1_EV), 160 (Kif24-3_EV), 153 (Kif24-3_KIF24). (D) The indicated Panc1 cells were cultured in serum-fed medium for 48 h, and their extracts were immunoblotted with anti-IFT88 and anti-KIF24-2 antibodies. β-Actin was used as a loading control. (E) The indicated Panc1 cells were cultured and immunostained as described in Fig 1B. The percentage of ciliated cells was determined. The average of four independent experiments is shown; >250 cells were scored each time. (F) The indicated Panc1 cells were cultured for 144 h, and the number of surviving cells was counted. The average of five independent experiments is shown. (G) The indicated Panc1 cells treated with distilled water (DW) or 0.5 mM ClHy were cultured in serum-fed medium for 48 h and immunostained as described in Fig 1B. The percentage of ciliated cells was determined. The average of three independent experiments is shown; >250 cells were scored each time. (H) The indicated Panc1 cells treated with DW or 0.5 mM ClHy were cultured for 144 h, and the number of surviving cells was counted. The average of five independent experiments is shown. (A, B, C, E, F, G, H) All data are shown as mean ± SD. Two-tailed t test. **P < 0.01; *P < 0.05. Source data are available for this figure.

Figure S2.. KIF24 mutation moderately promotes tumor formation in vivo.

(A, B, C) The indicated Panc1 cells were injected into nude mice. n = 10 (WT), 12 (Kif24-3). (A) The volume of tumor was measured every 2 wk. (B) Excised tumors were imaged. Scale bar, 10 mm. (C) The weight of excised tumors was measured. (D, E) Tumor slices were immunostained as described in Fig 1B. (D) Arrows indicate primary cilia. DNA was stained with Hoechst (blue). Scale bar, 10 μm. (E) The percentage of ciliated cells was determined. The average of three independent experiments is shown; >250 cells were scored each time. (A, C, E) All data are shown as mean ± SD. Two-tailed t test. **P < 0.01; *P < 0.05.

Figure S3.. Hedgehog signaling in KIF24-depleted Panc1 cells.

(A, B) The indicated Panc1 cells in serum-starved medium were treated with DMSO or 1 μM SAG for 48 h. Cells were immunostained with anti-ARL13B (green) and anti-GPR161 (red) antibodies. (A) DNA was stained with Hoechst (blue). Scale bar, 5 μm. (B) The percentage of cells with GPR161-positive cilia was determined. The average of three independent experiments is shown; >100 cells were scored each time. (C) The indicated cells were cultured in serum-fed medium for 48 h. Relative amounts of the indicated mRNA were determined using quantitative PCR. GAPDH was used as a control. Average of four independent experiments is shown. (B, C) All data are shown as mean ± SD. Two-tailed t test. NS, no significance.

Figure S4.. KIF24 localizes to centrosomes in Panc1 cells.

(A) Panc1 cells were immunostained with anti-centrin (green) and anti-KIF24 (red) antibodies. DNA was stained with Hoechst (blue). Scale bar, 10 μm. (B) The indicated Panc1 cells were immunostained with anti-centrin (green) and anti-KIF24 (red) antibodies. DNA was stained with Hoechst (blue). Cells in the metaphase were imaged. Scale bar, 5 μm.

Figure 3.. KIF24 depletion induces centrosome clustering in Panc1 cells.

(A, B) The indicated Panc1 cells were immunostained with anti-acetylated tubulin (green) and anti-phosphorylated AurA (pAurA) (red) antibodies. (A) DNA was stained with Hoechst (blue). Scale bar, 10 μm. (B) The percentage of cells with multipolar spindles in the metaphase was determined. The average of six independent experiments is shown; >100 cells were scored each time. (C, D) The indicated Panc1 cells were immunostained with anti-centrin (green) and anti-γ-tubulin (red) antibodies. (C) DNA was stained with Hoechst (blue). Scale bar, 10 μm. (D) The percentage of cells with pseudo bipolar or multipolar spindles in the metaphase was determined. The average of four independent experiments is shown; >100 cells were scored each time. (E, F) The indicated Panc1 cells were immunostained with an anti-phosphorylated Histone H3 (pHH3) (green) antibody. (E) DNA was stained with Hoechst (blue). Scale bar, 10 μm. (F) The percentage of cells with lagging chromosome in the anaphase was determined. The average of four independent experiments is shown; >100 cells were scored each time. (G, H) The indicated Panc1 cells were immunostained and quantified as described in Fig 3C and D. The average of five (G) or four (H) independent experiments is shown; >100 cells were scored each time. (I) Kif24-3 cells treated with DW or ClHy were cultured in serum-fed medium for 48 h. Cells were immunostained and quantified as described in Fig 3C and D. The average of three independent experiments is shown; >100 cells were scored each time. (B, D, F, G, H, I) All data are shown as mean ± SD. Two-tailed t test. **P < 0.01; *P < 0.05; NS, no significance.

Figure S5.. KIF24 depletion does not affect the centrosome number, centrosome fragmentation, and centriole duplication in Panc1 cells.

(A) The indicated PDAC cells were immunostained with anti-γ-tubulin and anti-pHH3 antibodies. The percentage of cells with 2> γ-tubulin dots in the interphase (pHH3-negative) and mitosis (pHH3-positive) was determined. The average of three to four independent experiments is shown; >100 cells were scored each time. (B, C) The indicated Panc1 cells were immunostained as described in Fig 3C. (B) The percentage of cells with γ-tubulin-positive and centrin-negative dot in the metaphase was determined. The average of four independent experiments is shown; >100 cells were scored each time. (C) The percentage of cells with >4 centrioles in the metaphase was determined. The average of four independent experiments is shown; >100 cells were scored each time. (D) The indicated Panc1 cells were cultured in serum-fed medium for 48 h. Cell extracts were immunoblotted with an anti-CP110 antibody. β-Actin was used as a loading control. (E, F) The indicated Panc1 cells were immunostained with anti-γ-tubulin (green) and anti-CENPJ/CPAP (red) antibodies. (E) DNA was stained with Hoechst (blue). Scale bar, 10 μm. (F) The quantified fluorescence intensity of CPAP at centrosome is shown. Interphase, n = 34 (Panc1_EV), 41 (Kif24-3_EV); mitosis, n = 24 (Panc1_EV), 24 (Kif24-3_EV). (A, B, C, D, F) All data are shown as mean ± SD. Two-tailed t test. NS, no significance. Source data are available for this figure.

Figure 4.. NEK2 phosphorylation or microtubule (MT)-depolymerizing activity dead mutants are sufficient for mitotic function of KIF24.

(A) The indicated Panc1 cells were cultured and immunoblotted as described in Fig 1A. (B) The indicated Panc1 cells were cultured and immunostained as described in Fig 1B. The percentage of ciliated cells was determined. The average of five independent experiments is shown; >250 cells were scored each time. (C) The indicated Panc1 cells were cultured, immunostained, and quantified as described in Fig 3C and D. The average of three to four independent experiments is shown; >100 cells were scored each time. (B, C) All data are shown as mean ± SD. Two-tailed t test. **P < 0.01; *P < 0.05. Source data are available for this figure.

Figure 5.. HSET/KIFC1 inhibition suppresses centrosome clustering induced by KIF24 depletion.

(A) The indicated Panc1 cells treated with DMSO or 50 μM CW069 for 4 h were cultured, immunostained, and quantified as described in Fig 3C and D. The average of five independent experiments is shown; >100 cells were scored each time. (B) The indicated Panc1 cells were immunostained with anti-HSET/KIFC1 (green) and anti-γ-tubulin (red) antibodies. DNA was stained with Hoechst (blue). Scale bar, 10 μm. (C) Panc1 cells treated with DMSO or 50 μM CW069 for 4 h were immunostained with anti-HSET/KIFC1 (green) and anti-KIF24 (red) antibodies. Arrows indicate KIF24 at spindles. DNA was stained with Hoechst (blue). Scale bar, 10 μm. (A) All data are shown as mean ± SD. Two-tailed t test. **P < 0.01; *P < 0.05.

Figure 6.. KIF24 depletion restores impaired mitotic events in Panc1 cells.

(A) Frames from live cell imaging of Panc1 or Kif24-3 cells stably expressing H2B-mCherry. Scale bar, 10 μm. (B) Time from nuclear envelope breakdown to the anaphase onset in indicated cells was measured. n = 119 (Panc1_H2B-mCherry), 135 (Kif24-3_H2B-mCherry). (C) The percentage of cells that entered and remained in mitosis for >180 min. The average of three independent experiments is shown; >40 cells were scored each time. (B, C) All data are shown as mean ± SD. Two-tailed t test. **P < 0.01; *P < 0.05.

Figure 7.. KIF24 depletion suppresses multipolar spindle formation and promotes cell growth in centrosome-amplified PDAC cells.

(A) The indicated PDAC cells were cultured and immunostained as described in Fig 1B. The percentage of ciliated cells was determined. The average of three (MiaPaCa2) or four (Hs766t) independent experiments is shown; >250 cells were scored each time. (B, C) The indicated PDAC cells were immunostained with anti-pHH3 (green) and anti-pAurA (red) antibodies. (B) DNA was stained with Hoechst (blue). Scale bar, 10 μm. (C) The percentage of cells with multipolar spindles in the metaphase was determined. The average of four (MiaPaCa2) or five (Hs766t) independent experiments is shown; >100 cells were scored each time. (D) The indicated cells were cultured for 96 h (MiaPaCa2) or 192 h (Hs766t), and the number of surviving cells was counted with a hemocytometer. The average of eight (MiaPaCa2) or six (Hs766t) independent experiments is shown. (A, C, D) All data are shown as mean ± SD. Two-tailed t test. **P < 0.01; *P < 0.05; NS, no significance.