CEP164 Deficiency Causes Hyperproliferation of Pancreatic Cancer Cells

Abstract

Primary cilia are hair-like projections that protrude from most mammalian cells and mediate various extracellular signaling pathways. Pancreatic ductal adenocarcinoma (PDAC) cells are known to lose their primary cilia, but the relevance of this phenomenon remains unclear. In this study, we generated PDAC-originated Panc1 cells devoid of primary cilia by mutating a centriolar protein, centrosomal protein 164 (CEP164), which is required for ciliogenesis. CEP164 depletion enhanced the clonogenicity of Panc1 cells, along with chemically induced elimination of primary cilia, suggesting that a lack of these organelles promotes PDAC cells proliferation. In addition, the loss of CEP164 altered the cell cycle progression irrespective of absence of primary cilia. We found that CEP164 was co-localized with the GLI2 transcription factor at the mother centriole and controlled its activation, thus inducing Cyclin D-CDK6 expression. Furthermore, CEP164-mutated Panc1 cells were significantly tolerant to KRAS depletion-dependent growth inhibition. This study suggests that CEP164 deficiency is advantageous for PDAC cells proliferation due to not only lack of ciliation but also cilia-independent GLI2-Cyclin D/CDK6 activation, and that CEP164 is a potential therapeutic target for PDAC.

Keywords: CEP164; PDAC; hedgehog; primary cilia; proliferation.

FIGURE 1

CEP164-mutated Panc1 cells show augmented colony-forming properties. (A,B) The indicated Panc1 cells were cultured in serum-fed (FBS+) or serum-starved medium (FBS–) for 48 h and immunostained with anti-glutamylated tubulin (GT335, green) and anti-Arl13b (red) antibodies. (A) Arrows indicate primary cilia. DNA was stained with Hoechst (blue). Scale bar, 10 μm. (B) The percentage of ciliated cells was determined. The average of three to four independent experiments is shown; >200 cells were scored each time. (C–E) The indicated cells were subjected to clonogenic assay. (C) Colonies were visualized with Crystal Violet and imaged. (D) The number of colonies was determined. The average of nine independent experiments is shown. (E) The OD at 595 nm of dissolved colonies was determined. The average of nine independent experiments is shown. (F,G) The indicated cells were subjected to soft agar assay. (F) Colonies were visualized with Crystal Violet and imaged. (G) The number of colonies was determined. The average of five independent experiments is shown. (B,D,E,G) All data are shown as mean ± SEM. **p < 0.01; *p < 0.05 compared with WT (two-tailed Student’s t-test).

FIGURE 2

Chemical de-ciliation enhances clonogenicity of Panc1 cells. (A–C) Panc1 cells treated with the indicated concentration of chloral hydrate (ClHy) for 14 days were subjected to clonogenic assay. (A) Colonies were visualized with Crystal Violet and imaged. (B) The number of colonies was determined. The average of three to four independent experiments is shown. (C) The OD at 595 nm of dissolved colonies was determined. The average of three to four independent experiments is shown. (D) Colonies in the clonogenic assay were immunostained with an anti-Arl13b antibody. The percentages of cells with primary cilia in each colony was determined and their averages are shown. Number of colonies analyzed = 40 (DW), 45 (0.1 mM ClHy), 56 (0.2 mM ClHy). (B–D) All data are shown as mean ± SEM. **p < 0.01; *p < 0.05 compared with distilled water (DW) (two-tailed Student’s t-test).

FIGURE 3

CEP164 depletion accelerates the cell cycle by elevation of Cyclin D-CDK6. (A) The indicated Panc1 cells were cultured in serum-fed medium for 48 h and analyzed using FACS. The proportion of cells at each cell cycle stage was determined. The average of three independent experiments is shown. (B,C) The indicated Panc1 cells were cultured in serum-fed medium for 48 h. (B) Relative amounts of the indicated mRNA were determined using quantitative PCR. GAPDH was used as a control. The average of four to eight independent experiments is shown. (C) Cell extracts were immunoblotted with indicated antibodies. β-Actin was used as a loading control. The amount of indicated proteins was quantified using ImageJ, and the relative values and GLI2^FL/GLI2^R ratio are shown below the image. (A,B) All data are shown as mean ± SEM. *p < 0.05 compared with Cep164-1 + EV (A) (Chi-squared test), compared with WT + EV and Cep164-1 + Cep164 (B) (two-tailed Student’s t-test). Uncropped images of western blots are shown in Supplementary Figure 5.

FIGURE 4

GLI2 dot disappears from the mother centriole in CEP164-mutated Panc1 cells. (A–C) The indicated cells were cultured in serum-fed medium for 48 h and immunostained with anti-glutamylated tubulin (green) and anti-GLI2 (red) antibodies. (A) DNA was stained with Hoechst (blue). Scale bar, 2.5 μm. (B) The percentage of cells with GLI2 dot at the non-ciliated centrosome was determined. The average of three independent experiments is shown; >100 cells were scored each time. (C) The quantified fluorescence intensity of GLI2 at centrosome is shown. n = 31 (WT + EV), 25 (Cep164-1 + EV), 35 (Cep164-1 + Cep164). (D) Panc1 cells were cultured in serum-fed medium for 48 h and immunostained with anti-CP110 (red), anti-CEP164 (blue), and anti-GLI2 (green) antibodies. Two representative images are shown. Scale bar, 2.5 μm. (B,C) All data are shown as mean ± SEM. **p < 0.01 compared with Cep164-1 + EV (B) (Chi-squared test), compared with Cep164-1 + EV (C) (two-tailed Student’s t-test).

FIGURE 5

Cep164-1 cells are moderately tolerant to growth delay provoked by KRAS depletion. (A–E) The indicated cells transiently transfected with siLucifearase (Luc) or siKras were cultured for 5 days. (A) Cell extracts were immunoblotted with an anti-KRAS antibody. β-Actin was used as a loading control. The amount of indicated proteins was quantified using ImageJ, and the relative values are shown below the image. (B) Cells were immunostained with an anti-Arl13b (green) antibody. DNA was stained with Hoechst (blue). White arrows indicate primary cilia. Scale bar, 10 μm. (C) The percentage of ciliated cells was determined. The average of three independent experiments is shown; >200 cells were scored each time. (D) Cells were subjected to Crystal Violet assay. Representative images are shown. Scale bar, 100 μm. (E) The OD at 595 nm of dissolved cells was determined. The average of five independent experiments is shown. (C,E) All data are shown as mean ± SEM. **p < 0.01; *p < 0.05 compared with siLuc (C) or WT (E) (two-tailed Student’s t-test). Uncropped images of western blots are shown in Supplementary Figure 5.