SCL/TAL1 Interrupting Locus Derepresses GLI1 from the Negative Control of Suppressor-of-Fused in Pancreatic Cancer Cell

Abstract

As a physically binding protein of GLI1 transcription factor, Suppressor-of-Fused (SUFU) has been placed in the center of negative regulation of Hedgehog (Hh) signaling. SUFU tethers GLI1 in cytoplasm, and in some circumstances, it moves into the nucleus in association with GLI1, leading to the suppression of GLI1 target gene expression by recruiting a corepressor complex. The activated transcriptional function of GLI1 is important for cellular proliferation in a variety of human cancers. However, it has not been revealed how GLI1 is derepressed from SUFU-mediated suppression. Here, we show SCL/TAL1 interrupting locus (SIL) product, a cytoplasmic protein overexpressed in pancreatic ductal adenocarcinoma (PDA), is responsible for the derepression of GLI1. We found SIL associated with the carboxyl terminus of SUFU, one of two distinct GLI1-binding domains, and this association was responsible for cytoplasmic tethering of SUFU. Overexpressed SIL attenuated SUFU-mediated cytoplasmic tethering and target gene suppression of GLI1. Knockdown of SIL in PDA cells conversely induced the nuclear accumulation of SUFU in association with GLI1 and the transcriptional suppression of GLI1 target genes. Importantly, we also showed that oncogenic K-RAS, and not Sonic hedgehog, enhanced the SIL association with the amino-terminus of SUFU, the other GLI1-binding domain that led to further increase of nuclear translocation of GLI1. These results uncover the role of SIL in derepressing GLI1 from the negative control of SUFU, which is a crucial step for activating Hh signaling in cancer cells. [Cancer Res 2008;68(19):7723-9]

GLI1 facilitates the migration and invasion of pancreatic cancer cells through MUC5AC-mediated attenuation of E-cadherin

“…GBS1mut and GBS2mut, mutants for putative GBSs (GBS1 and GBS2) in the MUC5AC promoter, were generated using the GeneTailor Site-Directed Mutagenesis System (Invitrogen, Carlsbad, CA, USA). Luciferase reporter assays were carried out using a Dual-Glo Luciferase Assay System (Promega) as described previously (Kasai et al, 2004(Kasai et al, , 2008. The following Stealth RNAi siRNAs (Invitrogen) were used to knock down GLI1 and GLI2: for GLI1 (siGLI1), 5 0 -AAUGCAAGGUCCCUC GUCCAAGCUG-3 0 , 5 0 -CAGCUUGGACGAGGGACCUUG CAUU-3 0 and for GLI2 (siGLI2), 5 0 -CAUAGAUGACCAC CUCAGCCUCCUG-3 0 , 5 0 -CAGGAGGCUGAGGUGGUCA UCUAUG-3 0 .…”

“…The following duplexes were used as controls: siControl-1, 5 0 -CGUACGCG GAAUACAACGATT-3 0 and 5 0 -UCGUUGUAUUCCGCG UACGTT-3 0 and siControl-2, 5 0 -CGCGAAACGAACGAAU AAATT-3 0 and 5 0 -UUUAUUCGUUCGUUUCGCGTT-3 0 . Immunoblot and semi-quantitative RT-PCR analyses were performed as described previously (Kasai et al, 2004(Kasai et al, , 2008. RNAs isolated from normal human tissues (the trachea, stomach, small and large intestine, gallbladder, pancreas and prostate: BioChain, Hayward, CA, USA) were combined, reverse-transcribed either with or without reverse transcriptase…”

“…GLI1 expression is induced in the pancreatic duct epithelium of low-grade PanIN, in which MU-C5AC is not yet expressed. Following either oncogenic activation of KRAS signaling (Pasca di Magliano et al, 2006;Kasai et al, 2008) or stimulation with Hedgehoginduced stromal factors (Yauch et al, 2008;NolanStevaux et al, 2009), GLI1 is derepressed from a pivotal suppressor of GLI proteins, Suppressor-of-Fused, and is consequently activated enough to induce the MUC5AC gene in high-grade PanIN. The abnormal lateral sorting of MUC5AC by an unknown mechanism abrogates the membrane localization and stabilization of E-cadherin, leading to the reduction or loss of membrane E-cadherin and the nuclear accumulation of b-catenin, which facilitates the migration and invasion of PDA cells.…”

“…Cells were then used for immunofluorescence staining as reported previously (Kasai et al, 2004(Kasai et al, , 2008. Z-stack confocal fluorescence images were obtained using an LSM710 fluorescence confocal microscope (Carl Zeiss MicroImaging, Goettingen, Germany) under a 40 Â water immersion lens.…”

Activation of the hedgehog-signaling pathway in human cancer and the clinical implications

“…Koga et al, 2008;Kasperczyk et al, 2009). Expression of Gli1 is reported to be regulated by TGFb, Ras, JUN, SCL/TAL1 and EWS-FLI1 oncoprotein (Ji et al, 2007;Kasai et al, 2008;Zwerner et al, 2008;Beauchamp et al, 2009;Laner-Plamberger et al, 2009;NolanStevaux et al, 2009). The interaction between PKC and hedgehog signaling varies depending on PKC isoforms and cell types.…”