Aurora kinase A inhibition plus Tumor Treating Fields suppress glioma cell proliferation in a cilium-independent manner.

Tumor Treating Fields (TTFields) extend the survival of glioblastoma (GBM) patients by interfering with a broad range of tumor cellular processes. Among these, TTFields disrupt primary cilia stability on GBM cells. Here we asked if concomitant treatment of TTFields with other agents that interfere with GBM ciliogenesis further suppress GBM cell proliferation in vitro. Aurora kinase A (AURKA) promotes both cilia disassembly and GBM growth. Inhibitors of AURKA, such as Alisertib, inhibit cilia disassembly and increase ciliary frequency in various cell types. However, we found that Alisertib treatment significantly reduced GBM cilia frequency in gliomaspheres across multiple patient derived cell lines, and in patient biopsies treated ex vivo. This effect appeared glioma cell-specific as it did not reduce normal neuronal or glial cilia frequencies. Alisertib-mediated depletion of glioma cilia appears specific to AURKA and not AURKB inhibition, and attributable in part to autophagy pathway activation. Treatment of two different GBM patient-derived cell lines with TTFields and Alisertib resulted in a significant reduction in cell proliferation compared to either treatment alone. However, this effect was not cilia-dependent as the combined treatment reduced proliferation in cilia-depleted cell lines lacking, ARL13B, or U87MG cells which are naturally devoid of ARL13B+ cilia. Thus, Alisertib-mediated effects on glioma cilia may be a useful biomarker of drug efficacy within tumor tissue. Considering Alisertib can cross the blood brain barrier and inhibit intracranial growth, our data warrant future studies to explore whether concomitant Alisertib and TTFields exposure prolongs survival of brain tumor-bearing animals in vivo.

Increasing Ciliary ARL13B Expression Drives Active and Inhibitor-Resistant Smoothened and GLI into Glioma Primary Cilia.

ADP-ribosylation factor-like protein 13B (ARL13B), a regulatory GTPase and guanine exchange factor (GEF), enriches in primary cilia and promotes tumorigenesis in part by regulating Smoothened (SMO), GLI, and Sonic Hedgehog (SHH) signaling. Gliomas with increased ARL13B, SMO, and GLI2 expression are more aggressive, but the relationship to cilia is unclear. Previous studies have showed that increasing ARL13B in glioblastoma cells promoted ciliary SMO accumulation, independent of exogenous SHH addition. Here, we show that SMO accumulation is due to increased ciliary, but not extraciliary, ARL13B. Increasing ARL13B expression promotes the accumulation of both activated SMO and GLI2 in glioma cilia. ARL13B-driven increases in ciliary SMO and GLI2 are resistant to SMO inhibitors, GDC-0449, and cyclopamine. Surprisingly, ARL13B-induced changes in ciliary SMO/GLI2 did not correlate with canonical changes in downstream SHH pathway genes. However, glioma cell lines whose cilia overexpress WT but not guanine exchange factor-deficient ARL13B, display reduced INPP5e, a ciliary membrane component whose depletion may favor SMO/GLI2 enrichment. Glioma cells overexpressing ARL13B also display reduced ciliary intraflagellar transport 88 (IFT88), suggesting that altered retrograde transport could further promote SMO/GLI accumulation. Collectively, our data suggest that factors increasing ARL13B expression in glioma cells may promote both changes in ciliary membrane characteristics and IFT proteins, leading to the accumulation of drug-resistant SMO and GLI. The downstream targets and consequences of these ciliary changes require further investigation.

Tumor Treating Fields Suppression of Ciliogenesis Enhances Temozolomide Toxicity.

Tumor Treating Fields (TTFields) are low-intensity, alternating intermediate-frequency (200 kHz) electrical fields that extend survival of glioblastoma patients receiving maintenance temozolomide (TMZ) chemotherapy. How TTFields exert efficacy on cancer over normal cells or interact with TMZ is unclear. Primary cilia are microtubule-based organelles triggered by extracellular ligands, mechanical and electrical field stimulation and are capable of promoting cancer growth and TMZ chemoresistance. We found in both low- and high-grade patient glioma cell lines that TTFields ablated cilia within 24 h. Halting TTFields treatment led to recovered frequencies of elongated cilia. Cilia on normal primary astrocytes, neurons, and multiciliated/ependymal cells were less affected by TTFields. The TTFields-mediated loss of glioma cilia was partially rescued by chloroquine pretreatment, suggesting the effect is in part due to autophagy activation. We also observed death of ciliated cells during TTFields by live imaging. Notably, TMZ and TTFields have opposing effects on glioma ciliogenesis. TMZ-induced stimulation of ciliogenesis in both adherent cells and gliomaspheres was blocked by TTFields. Surprisingly, the inhibitory effects of TTFields and TMZ on tumor cell recurrence are linked to the relative timing of TMZ exposure to TTFields and ARL13B+ cilia. Finally, TTFields disrupted cilia in patient tumors treated ex vivo. Our findings suggest that the efficacy of TTFields may depend on the degree of tumor ciliogenesis and relative timing of TMZ treatment.