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. 2016 Feb 9;7(6):7029-43.
doi: 10.18632/oncotarget.6854.

Disruption of KIF3A in patient-derived glioblastoma cells: effects on ciliogenesis, hedgehog sensitivity, and tumorigenesis

Lan B Hoang-Minh  1   2 , Loic P Deleyrolle  3   2 , Dorit Siebzehnrubl  1 , George Ugartemendia  1 , Hunter Futch  3 , Benjamin Griffith  3 , Joshua J Breunig  4   5 , Gabriel De Leon  3   6 , Duane A Mitchell  3   2   6 , Susan Semple-Rowland  1 , Brent A Reynolds  3   2 , Matthew R Sarkisian  1   2
Affiliations

Disruption of KIF3A in patient-derived glioblastoma cells: effects on ciliogenesis, hedgehog sensitivity, and tumorigenesis

Lan B Hoang-Minh et al. Oncotarget. .

Abstract

KIF3A, a component of the kinesin-2 motor, is necessary for the progression of diverse tumor types. This is partly due to its role in regulating ciliogenesis and cell responsiveness to sonic hedgehog (SHH). Notably, primary cilia have been detected in human glioblastoma multiforme (GBM) tumor biopsies and derived cell lines. Here, we asked whether disrupting KIF3A in GBM cells affected ciliogenesis, in vitro growth and responsiveness to SHH, or tumorigenic behavior in vivo. We used a lentiviral vector to create three patient-derived GBM cell lines expressing a dominant negative, motorless form of Kif3a (dnKif3a). In all unmodified lines, we found that most GBM cells were capable of producing ciliated progeny and that dnKif3a expression in these cells ablated ciliogenesis. Interestingly, unmodified and dnKif3a-expressing cell lines displayed differential sensitivities and pathway activation to SHH and variable tumor-associated survival following mouse xenografts. In one cell line, SHH-induced cell proliferation was prevented in vitro by either expressing dnKif3a or inhibiting SMO signaling using cyclopamine, and the survival times of mice implanted with dnKif3a-expressing cells were increased. In a second line, expression of dnKif3a increased the cells' baseline proliferation while, surprisingly, sensitizing them to SHH-induced cell death. The survival times of mice implanted with these dnKif3a-expressing cells were decreased. Finally, expression of dnKif3a in a third cell line had no effect on cell proliferation, SHH sensitivity, or mouse survival times. These findings indicate that KIF3A is essential for GBM cell ciliogenesis, but its role in modulating GBM cell behavior is highly variable.

Keywords: brain tumor; cilium; intraflagellar transport; kinesin-2; sonic hedgehog.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. A majority of isolated patient-derived GBM cells produce ciliated progeny
(A) Spheres of Line 0 (L0) and S3 GBM cells were dissociated, sorted as single cells into 96-well plates, and further expanded onto coverslips in 24-well plates prior to immunostaining for cilia markers after 24 hours. (B) Confocal maximum projection images of L0 clones (1 and 15) immunostained for PCM1 (green) and acetylated alpha-tubulin (aa-tubulin; red). The merged images (right panels) show aa-tubulin-positive axonemes (arrows) projecting from PCM1-positive basal bodies (arrowheads) in Clone 15 but not in Clone 1. (C) Percentage of all clones in L0 or S3 cell lines that gave rise to ciliated progeny. (D) Percentage of L0 or S3 cells with aa-tubulin-positive cilia derived from individual clones. Scale bar in B = 10 μm.
Figure 2
Figure 2. The expression of dnKif3a potently inhibits cilia formation in three GBM cell lines
L0, S2 and S3 cell lines were infected with lentiviral vectors encoding either mCherry alone or mCherry and dominant negative Kif3a (dnKif3a) and their cilia examined using immunocytochemistry and EM. Next, we used FAC-sorting to select mCherry-positive L0, S2, and S3 cells, expanded the sorted cells, and assessed whether they were able to form cilia using ultrastructural and immunocytochemical techniques. (A) Confocal image shows L0 cells (prior to sorting) with aa-tubulin-positive cilia on infected mCherry-positive cells (arrows) and non-infected mCherry-negative cells (arrowhead). (A') Sorted mCherry-positive cells were fixed and analyzed by EM. The example shows a cilium with a docked basal body (arrow) and a long ciliary axoneme (arrowheads) projecting outside the cell. (B) L0 cells (prior to sorting) revealed aa-tubulin-positive cilia but only on non-infected (mCherry-negative) cells (arrowheads) and not on infected mCherry and dnKif3a-positive cells. (B', B”) By EM, sorted L0 mCherry and dnKif3a-positive cells revealed basal bodies (arrows) that lacked a clear axoneme (B'; arrowhead) or displayed an abnormally assembled/poorly formed axoneme (B”; arrowhead). (C) Percentage (+/− SEM) of aa-tubulin-positive ciliated cells among sorted mCherry-positive and sorted mCherry and dnKif3a-positive cells for the indicated cell lines. (D) Endogenous KIF3A levels are reduced in L0, S2 and S3 GBM cell lysates after dnKif3a expression. Thirty μg of total protein lysates from L0, S2 and S3 cells expressing mCherry or mCherry and dnKif3a were separated by SDS-PAGE and western blotted with an antibody against human KIF3A protein. Although KIF3A (∼ 85 kDa) was detected in all groups, lysates from mCherry and dnKif3a-expressing cells consistently displayed reduced KIF3A levels compared to those expressing mCherry alone. We also observed a smaller band (∼ 40 kDa, arrowhead) in lanes with dnKif3a-expressing cell lysates, which might either represent degraded endogenous KIF3A or possibly a sequence associated with the expression of dnKif3a. Δ-actin was run as a loading control. Scale bars for A and B = 10 μm; A' = 500 nm; B' and B” = 250 nm. ***p<0.005 (Student's t-test).
Figure 3
Figure 3. The effects of dnKif3a expression and SHH on GBM cell proliferation are cell-line dependent
Mean cell numbers (+/− SEM) per well for L0 (A), S2 (B) and S3 (C) mCherry-positive and mCherry and dnKif3a-positive cells five days after exposure to vehicle or recombinant human SHH (1 μg/ml), with or without prior addition of cyclopamine (1 μM). Groups were compared using a one-way ANOVA followed by Tukey's posthoc analysis. *p<0.05, ***p<0.005.
Figure 4
Figure 4. SHH treatment induces changes in SHH signaling pathway gene expression in L0, but not S2 and S3 cell lines
(A-C) The mRNA expression levels of GLI1, GLI2, GLI3, PTCH, SMO, SHH, and SUFU were estimated by real-time qPCR. Increases in SHH pathway gene expression were observed in the L0 (A) but not S2 (B) or S3 (C) cell line (n = 3 biological replicates/gene). The expression of each target gene was quantified relative to β-actin mRNA. Data are expressed as means ± SEM. *p<0.05, **p<0.01, ***p<0.005 (Student's t-test).
Figure 5
Figure 5. The relationship between dnKif3a expression and mouse survival following intracranial xenograft is cell-line dependent
(A-C) Kaplan-Meier curves for mice xenografted with mCherry-positive control (red) or mCherry and dnKif3a-positive (blue) L0 (A), S2 (B) and S3 (C) cells. n = number of mice per group. (D-F) Sections through the mCherry-positive primary tumor mass immunostained for Arl13b, a marker of primary cilia. In L0 (D), S2 (E) and S3 (F) cell lines, we detected numerous Arl13b-positive cilia (arrowheads) in mCherry-positive control tumors, but not in tumors comprised of cells expressing mCherry and dnKif3a. Boxed regions in D-F are enlarged in the right panel insets to show that, in conjunction with staining for gamma-tubulin (gtub), Arl13b-positive cilia extend from gamma-tubulin-positive basal bodies (red) in mCherry control but not mCherry and dnKif3a-positive cells. Scale bars in D-F = 10 μm.
Figure 6
Figure 6. Ciliated, SMO-positive L0 cells are found in sites distal to the tumor mass
(A) Image of mCherry and Arl13b-positive cells (arrowheads) in satellite growths that formed outside of the primary tumor core in L0 control cell-derived tumors. (C) mCherry-positive cell displaying a migratory profile with a short trailing process (arrow) and long leading process (arrowheads). (B, D) Gamma-tubulin (gTub) (red) was substituted for mCherry in A and C, respectively. Arrows point to Arl13b-positive cilia with G-tubulin-positive basal bodies enlarged in insets. (E) Example of an mCherry-positive cell observed away from the primary tumor mass displaying gamma-tubulin positive basal bodies (arrowheads) and harboring a SMO-positive cilium (arrows). Scale bars for A and C = 10 μm; E = 5 μm.
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