Abstract 995: Integrin-Linked Kinase localizes in the primary cilia to mediate Hedgehog signaling

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The Hedgehog (Hh) signaling pathway is a highly conserved system for regulating metazoan development and cell fate. Integrin linked kinase (ILK) is required for proliferation of Hh-dependent neurons in the mammalian cerebellum, which also give rise to medulloblastoma, the most common childhood brain tumour. Sonic Hedgehog (SHh) binds to the Ptch transmembrane receptor, derepressing the Smo receptor to activate Hh signaling and key downstream targets such as Gli transcription factors. We find that SHh-induced osteogenic differentiation of C3H10T1/2 cells is blocked (>90% inhibition) by a small molecule ILK inhibitor, or by ILK-specific RNAi. SHh induces alkaline phosphatase (AP) activity in control 10T1/2 cells, but not cells transfected with ILK siRNA (>75% knockdown). Moreover we find that the small molecule ILK inhibitor suppresses SHh-induced differentiation in a dose-dependent manner, as effectively and to the same extent as the well-characterized selective Hh inhibitor, cyclopamine. Activation of Smo with the agonist, SAG, induces ILK-dependent AP induction in these cells, suggesting ILK acts downstream of Smo in the Hh pathway. SHh induction of the Gli1 transcription factor is also blocked by inhibiting ILK, suggesting ILK acts upstream of direct Hh targets. ILK inhibitor or siRNA suppresses constitutively activated Gli1 in medulloblastoma cells, derived from Ptch+/-:p53 compound heterozygote mice. In support of ILK involvment in Hh signaling, we have discovered a novel localization of ILK that indicates spatial proximity to Hh signaling molecules. The primary cilium is a microtubule-based organelle that is required for Hedgehog signaling, and we now find that ILK localizes in this structure. Using a selective ciliary marker, acetylated α-tubulin, we find that ILK localizes in the primary cilium along with components of the Hh pathway. Inhibition of ILK does not inhibit cilia formation, indicating that ILK is not involved in ciliogenesis. IFT proteins (eg. kinesin II motor subunit Kif3a), regulate both the assembly of, and the entry of Hh signaling proteins to, the cilium. Knockdown of Kif3a inhibits ciliogenesis and efficiently blocks SHh signaling, strongly suggesting the ciliary pool of ILK is critical for Hh signaling. Our work points to ILK as a novel therapeutic target in Hh-activated cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 995.