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. 2019 Apr;110(4):1306-1316.
doi: 10.1111/cas.13970. Epub 2019 Feb 28.

Intraflagellar transport 20 promotes collective cancer cell invasion by regulating polarized organization of Golgi-associated microtubules

Tomoaki Aoki  1   2 , Michiru Nishita  1 , Junya Sonoda  1 , Taro Ikeda  1   2 , Yoshihiro Kakeji  2 , Yasuhiro Minami  1
Affiliations

Intraflagellar transport 20 promotes collective cancer cell invasion by regulating polarized organization of Golgi-associated microtubules

Tomoaki Aoki et al. Cancer Sci. 2019 Apr.

Abstract

Collective invasion is an important strategy of cancers of epithelial origin, including colorectal cancer (CRC), to infiltrate efficiently into local tissues as collective cell groups. Within the groups, cells at the invasive front, called leader cells, are highly polarized and motile, thereby providing the migratory traction that guides the follower cells. However, its underlying mechanisms remain unclear. We have previously shown that signaling emanating from the receptor tyrosine kinase Ror2 can promote invasion of human osteosarcoma cells and that intraflagellar transport 20 (IFT20) mediates its signaling to regulate Golgi structure and transport. Herein, we investigated the role of Ror2 and IFT20 in collective invasion of CRC cells, where Ror2 expression is either silenced or nonsilenced. We show by cell biological analyses that IFT20 promotes collective invasion of CRC cells, irrespective of expression and function of Ror2. Intraflagellar transport 20 is required for organization of Golgi-associated, stabilized microtubules, oriented toward the direction of invasion in leader cells. Our results also indicate that IFT20 promotes reorientation of the Golgi apparatus toward the front side of leader cells. Live cell imaging of the microtubule plus-end binding protein EB1 revealed that IFT20 is required for continuous polarized microtubule growth in leader cells. These results indicate that IFT20 plays an important role in collective invasion of CRC cells by regulating organization of Golgi-associated, stabilized microtubules and Golgi polarity in leader cells.

Keywords: IFT20; Ror2; collective invasion; colorectal cancer; noncentrosomal microtubule.

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Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
Suppressed expression of intraflagellar transport 20 (IFT20) impairs invasive migration of Ror2‐expressing colorectal cancer (CRC) cells. A, Expression levels of Ror2 and IFT20 in the CRC cell lines DLD1, HCT116, and SW480. Whole cell lysates from the respective CRC cells were analyzed by western blotting with Abs against the indicated proteins. B‐E, Decreased RNA (B,D) and protein (C,E) levels of IFT20 in HCT116 (B,C), but not SW480 cells (D,E), transfected with Ror2 siRNA. Cells were transfected with control (Ctrl), Ror2, or IFT20 siRNA and analyzed by real‐time PCR (B,D) and western blotting (C,E) to monitor amounts of the indicated RNAs and proteins, respectively. F‐I, HCT116 (F,G) and SW480 (H,I) cells were transfected with the indicated siRNAs and subjected to 2‐D invasion assay. Representative phase contrast images before (0 h) and after incubation for 18 h are shown (F,H). Scale bar, 500 μm. Invasion ratios of the respective cells after incubation for 18 h were determined (G,I). Data are expressed as mean ± SD (n = 3). *P < .05, t test
Figure 2
Figure 2
Intraflagellar transport 20 (IFT20) is required for accumulation of acetylated microtubules during collective invasion. A,B, Suppressed mRNA (A) and protein (B) expression of IFT20 in DLD1 cells transfected with siRNAs against IFT20. C,D, siRNA‐transfected DLD1 cells were analyzed by 2‐D invasion assay. Representative phase contrast images of cells before (0 h) and after incubation for 18 h are shown (C). Yellow lines indicate edges of cell layers. Scale bar, 500 μm. Invasion ratios of the respective cells after incubation for 0, 6, 12, and 18 h were determined 2(D). Data are expressed as mean ± SD (n = 3). *P < .05; **P < .005, t test. E, Relative viable cell number of siRNA‐transfected DLD1 cells at 0 and 18 h in 2‐D invasion assay was measured using WST8. Data are expressed as mean ± SD (n = 3). F, siRNA‐transfected DLD1 cells were analyzed by Transwell invasion assay. Cells invaded to the lower surface of the Transwell membranes were counted. Data are expressed as mean ± SD (n = 3). *P < .05, t test. G,H, siRNA‐transfected DLD1 cells were subjected to 2‐D invasion assay for 18 h. Cells were stained with Abs against α‐tubulin or acetylated (Ac)‐tubulin (green) and counterstained with DAPI (blue). G, Representative confocal images of cells. Scale bar, 20 μm. H, Fluorescence intensities of α‐tubulin and Ac‐tubulin in leader cells, normalized by those in follower cells, were quantified, and the ratio of fluorescence intensities of Ac‐tubulin and α‐tubulin were determined. Data are expressed as mean ± SD (n = 3, >30 cells/experiment). *P < .05; **P < .005, t test
Figure 3
Figure 3
Intraflagellar transport 20 (IFT20) is required for organization of Golgi‐associated microtubules during collective invasion. A, DLD1 cells transfected with the indicated siRNAs were subjected to 2‐D invasion assay for 18 h. Cells were stained with Abs against acetylated (Ac)‐tubulin (green) and GM130 (red) and analyzed by super‐resolution microscopy. Representative leader cells are shown with the leading edge to the right. Lower panels show enlarged views of the boxed region in the upper panels. Scale bar, 10 μm (upper panels) and 1 μm (lower panels). Note that acetylated microtubules are highly organized and associated with the Golgi apparatus in control (si‐Ctrl) cells, but poorly organized in si‐IFT20 cells. B, Fluorescence intensities of Ac‐tubulin surrounding the Golgi in leader cells were quantified. Data are expressed as mean ± SD of 12 cells. ***P < .001, t test
Figure 4
Figure 4
Intraflagellar transport 20 (IFT20) is required for polarization of the Golgi toward the leading edge during collective invasion of colorectal cancer cells. A, DLD1 cells transfected with the indicated siRNAs were subjected to 2‐D invasion assay for 18 h. Cells were stained with Abs against acetylated (Ac)‐tubulin (green) and GM130 (red), and counterstained with DAPI (blue). Arrows indicate the Golgi facing to the leading edge in leader cells. Scale bar, 10 μm. B, Percentages of leader and follower cells, in which the Golgi apparatus was detected within the 120° sector emerging from the center of the nucleus and facing toward the leading edge, were measured. Data are expressed as mean ± SD (n = 3). *P < .05; **P < .005, t test. si‐Ctrl, negative control siRNA
Figure 5
Figure 5
Suppressed expression of intraflagellar transport 20 (IFT20) impairs polarized and continuous microtubule growth during collective invasion of colorectal cancer cells. DLD1 cells expressing EB1‐GFP were transfected with the indicated siRNAs and subjected to 2‐D invasion assay. After 16 h of incubation, movement of EB1‐GFP in leader cells was analyzed by time‐lapse fluorescence imaging [Link]and tracking programs. A, Circular histograms showing the percentages of EB1‐GFP comets whose moving direction was within each of 36 equal sectors (10°). 0° indicates the direction perpendicular to the leading edge of the original cell sheet. The analysis included 2300‐2500 comets from 15 cells per each condition. B,C, Quantification of pause frequency (B) and moving speed (C) of EB1‐GFP comets. We defined a pause as an absence of movement in either direction for at least 0.9 s. Data are presented as box‐and‐whisker plots. n = 300‐520 comets; ***P < .001, t test. si‐Ctrl, negative control siRNA
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