A novel protein encoded by circular SMO RNA is essential for Hedgehog signaling activation and glioblastoma tumorigenicity

Abstract

Background: Aberrant activation of the Hedgehog pathway drives tumorigenesis of many cancers, including glioblastoma. However, the sensitization mechanism of the G protein-coupled-like receptor smoothened (SMO), a key component of Hedgehog signaling, remains largely unknown.

Results: In this study, we describe a novel protein SMO-193a.a. that is essential for Hedgehog signaling activation in glioblastoma. Encoded by circular SMO (circ-SMO), SMO-193a.a. is required for sonic hedgehog (Shh) induced SMO activation, via interacting with SMO, enhancing SMO cholesterol modification, and releasing SMO from the inhibition of patched transmembrane receptors. Deprivation of SMO-193a.a. in brain cancer stem cells attenuates Hedgehog signaling intensity and suppresses self-renewal, proliferation in vitro, and tumorigenicity in vivo. Moreover, circ-SMO/SMO-193a.a. is positively regulated by FUS, a direct transcriptional target of Gli1. Shh/Gli1/FUS/SMO-193a.a. form a positive feedback loop to sustain Hedgehog signaling activation in glioblastoma. Clinically, SMO-193a.a. is more specifically expressed in glioblastoma than SMO and is relevant to Gli1 expression. Higher expression of SMO-193a.a. predicts worse overall survival of glioblastoma patients, indicating its prognostic value.

Conclusions: Our study reveals that SMO-193a.a., a novel protein encoded by circular SMO, is critical for Hedgehog signaling, drives glioblastoma tumorigenesis and is a novel target for glioblastoma treatment.

Keywords: Brain cancer stem cells; Circular RNA; Glioblastoma; Hedgehog pathway; Novel protein.

Fig. 1

circ-SMO is highly expressed in CSCs and GBM. a Gli1 mRNA level in NHA, established GBM cell lines and CSCs. b A total of 12 pairs of GBM and NB samples were subjected to RNA-seq and CIRIquant analysis. Left, differentially expressed circRNAs (p < 0.01 and fold change > 2) with circBase annotation. In total, 1791 circRNAs were upregulated in GBM; 2299 circRNAs were downregulated in GBM. Right, top five highly expressed circRNAs are listed between GBM and NB. c Upper, exons 3–6 of SMO formed circ-SMO. Lower left, PCR products of linear SMO and circ-SMO using convergent or divergent primers. Lower right, Sanger sequencing of circ-SMO junction sequences. d Circ-SMO characters in 3691 CSC. Upper, qPCR of linear or circ-SMO from oligo dT primers and random primers reversely transcripted cDNA. Middle, qPCR of linear SMO or circ-SMO after RNase R treatment. Lower, half-life of linear SMO or circ-SMO. e Left, Northern blotting of circ-SMO and SMO mRNA transcripts by hybridization with exon 4 probes in the absence or presence of RNase R treatment in 456 and 3691 CSCs. Right, junction-specific probe was used to evaluate circ-SMO levels with or without circ-SMO overexpression in indicated cells. Illustration shows the targets of circ-SMO probe, circ-SMO junction shRNAs (referred as sh1 and sh2), and scramble shRNA. f Fluorescence in situ hybridization (FISH) of circ-SMO in 3691 CSC with indicated modifications. Bars, 10 μm. g Cell fraction qPCR in 3691 CSC. β-actin and U6 were used as cytoplasmic or nuclear markers. h Relative expression level of circ-SMO in different cell lines. i Left, relative expression level of circ-SMO in 86 GBM patients and their paired adjacent NB tissues. Right, Survival analysis of 86 GBM patients based on circ-SMO expression. The median score of relative expression levels in tumor tissues based on RT-qPCR was used as the cutoff to define “high” or “low” Circ-SMO expression. MST, median survival time. Lines show the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. In a, c, d, f, g, h, i, Data are representative from at least three experiments with similar results

Fig. 2

Circ-SMO encodes SMO-193a.a. a 293T cells transfected with circ-SMO or circ-SMO noATG plasmid were subjected to polysome profiling assay. Left, Representative polysome profile. Cell lysates were fractionated to collect non-ribosome fractions (N), monosome (M), light polysome (L), and heavy polysome (H) by 5–50% sucrose gradient centrifugation. Dashed lines indicate collected fractions. Right, Detection of circ-SMO and SMO mRNA in indicated fractions by RT-qPCR in 293T cells with indicated modifications. b Left, Predicted start and stop codon of ORF in circ-SMO. Right, Predicted amino acid sequences of SMO-193a.a. and antigen sequences for SMO-193a.a. antibody. c Identification and activity test of internal ribosomal entry site (IRES) in circ-SMO using circular vector-based luciferase reporter assay. EMCV-IRES was positive control. d Immunoblot (IB) and mass spectra (MS) identification of SMO-193a.a. in 373 GBM cells with circ-SMO overexpression (OV) and in 3691 CSC. e Upper, Illustration of SMO shRNAs and circ-SMO shRNAs. Lower, SMO and SMO-193a.a. expressions were detected using SMO-193a.a. polyclonal antibody in 3691 CSC treated with indicated shRNAs. f Left, Illustration of endogenous circ-SMO, circ-SMO overexpression vector, splicing donor site mutant vector (mutSD), circularization frame deletion vector (circ-frame Del), circ-SMO-3XFlag vector, and linearized SMO-193a.a.-3XFlag vector. Right, IB of cells OV with above described different vectors using anti-SMO-193a.a. antibody or Flag antibody. g. Left, Representative immunofluorescence (IF) images of cells OV with indicated vectors and stained with anti-Flag antibody. Bars, 5 μm. Right, Live image of SMO-193a.a.-mCherry in 373 GBM cells. Bars, 5 μm. h Left, IB of several established cell lines using anti-SMO-193a.a. antibody. Right, IB of seven random selected GBM samples and adjacent NB using anti-SMO-193a.a. antibody. i Upper, semi-quantification of SMO-193a.a. expression based on immunoblot band intensity relative to beta-tubulin in a previously described cohort of 86 GBM samples and paired NB. Lower, Survival analysis was conducted based on SMO-193a.a. expression in 86 GBM patients. The median score of relative expression levels in tumor tissues based on semi-quantification was used as the cutoff to define “high” or “low” SMO-193a.a. expression. MST, median survival time. Lines show the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. Data are representative from at least three experiments with similar results

Fig. 3

SMO-193a.a. maintains self-renewal of CSCs. a IB of SMO-193a.a. in 456, 3691 CSCs stably knocking down (KD) circ-SMO and re-expressed circ-SMO or SMO-193a.a. linearized vector. b qPCR of circ-SMO in in 456, 3691 CSCs KD circ-SMO and re-expressed circ-SMO or SMO-193a.a. linearized vector. c Limited dilution assay (LDA) of 456, 3691 CSCs with indicated modifications. Stem cell frequencies were calculated. Lines show the estimate values, upper/lower limits of confidence intervals. d Cell proliferation of 456, 3691 CSCs with indicated modifications. e EdU incorporation assay of 456, 3691 CSCs with indicated modifications. f Stemness markers including Sox2, Oct4, Nestin, and Nanog; differentiation markers GFAP and Tuj-1 were determined by IB in 456, 3691 CSCs with indicated modifications. g Upper, Illustration of circ-SMO mutant vector. An A was inserted after the start codon of SMO-193a.a. to compromise the ORF. Lower, qPCR and IB were used to verify circ-SMO mutant vector. h LDA of 456, 3691 CSCs with indicated modifications. Stem cell frequencies were calculated. Lines show the estimate values, upper/lower limits of confidence intervals. i Cell proliferation of 456, 3691 CSCs with indicated modifications. j EdU incorporation assay of 456, 3691 CSCs with indicated modifications. Lines show the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. In a to j, data are representative from at least three experiments with similar results

Fig. 4

SMO-193a.a. activates HH signaling in CSCs. a RNA-seq and Gene Set Enrichment Analysis (GSEA) of 456 and 3691 CSCs treated with circ-SMO scramble shRNA or sh1. b Gli1 binding site luciferase assay of 3691 and 387 CSCs with indicated modifications. c SMO and SMO-193a.a., SMO mRNA, and circ-SMO, as well as Gli1, C-Myc, and CCND1 mRNA expression were determined by IB or qPCR in 3691 CSC with indicated modifications. d SMO and SMO-193a.a., SMO mRNA, and circ-SMO, as well as Gli1, C-Myc, and CCND1 mRNA expression were determined by IB or qPCR in 387 CSC with indicated modifications. e IB of Gli1, c-Myc, and cyclin D1 expression in 3691, 387 CSCs with indicated modifications. f HH signaling agonist SAG and HH signaling inhibitor vismodegib were used in 3691 CSC with circ-SMO KD or in 387 CSC with circ-SMO OV. IB was used to determine the expression of indicated proteins. g Gli1 was OV in 456 and 3691 CSCs with circ-SMO KD. SMO-193a.a. and Gli1 level were determined by IB. h LDA was performed in 456 and 3691 CSCs with circ-SMO KD and Gli1 OV. Stem cell frequencies were calculated. Lines show the estimate values, upper/lower limits of confidence intervals. i Cell proliferation assay was performed in 456 and 3691 CSCs with circ-SMO KD and Gli1 OV. Lines show the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. In b to i, data are representative from at least three experiments with similar results

Fig. 5

SMO-193a.a. interacts with SMO, promotes SMO activation, and is required for HH-induced SMO de-repression. a In vivo IP between SMO and SMO-193a.a. in 456 and 3691 CSCs. b In vitro IP using purified SMO and SMO-193a.a.. c Colocalization of SMO and SMO-193a.a.-mCherry in 387 CSC. Bars, 2 μm. d p-SMO and SMO were determined in 3691 and 387 CSCs with indicated modifications. e Upper left, Illustration of SMO-193a.a. 5 transmembrane helix structures and SMO 7 transmembrane helix structures. Upper right, Illustration of full-length and truncated SMO-HA-tagged plasmids. Lower, Full-length and truncated SMO as indicated were co-IP with SMO-193a.a. in 373 GBM cells with SMO-193a.a.-3XFlag and HA-SMO domains OV. f Upper, Illustration of cholesterol labeling experiments. A specific nucleotide sequence was labeled with cholesterol and added to 387 CSC with SMO-193a.a. OV or ctrl OV, or 3691 CSC with circ-SMO KD or ctrl KD. Lower, Cholesterol modified SMO or SMO-193a.a. were determined by IP followed qPCR. g 2.5μg/ml Shh was added to 456 and 3691 CSCs with indicated modifications. IB was used to determine the expression level of Gli1, p-SMO, and SMO at indicated timepoints. sh1 + 2 defines circ-SMO stable knocking down cells generated by using 1:1 cocktail mixture of sh1 and sh2. h Different concentrations of Shh was added to 456 and 3691 CSCs with indicated modifications. IB was used to determine the expression level of Gli1, p-SMO, and SMO after 24 h. Lines show the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. Data are representative from at least three experiments with similar results

Fig. 6

SMO-193a.a. is a downstream target of HH signaling and in vivo effects of SMO-193a.a alteration. a Left, circ-SMO expression in 456 and 3691 CSCs treated with Shh. Right, IB of SMO-193a.a. and Gli1 in 456 and 3691 CSCs treated with Shh. b IB of SMO-193a.a. and Gli1 in 456 and 3691 CSCs with FUS KD. c Luciferase assay of wild type or Gli1 binding site mutated FUS promoter activities after Shh stimulation. d ChIP assay of FUS promoter sequences using Gli1 antibody in 456 and 3691 CSCs. Bcl2 was used as positive control. e IB and qPCR of FUS protein and mRNA levels in 456 and 3691 CSCs with Gli1 KD. f FUS and Gli1 protein level in 9 randomly selected GBM samples. g Upper, Representative images of in vivo tumorigenicity assay collected at day 25(456 CSC) and day 30 (3691 CSC) post-implantation using 456 and 3691 CSCs with indicated modifications. Each group contains 5 mice. Lower, Representative images of immunohistochemistry (IHC) of Gli1 expression in above mice. h Survival analysis of in vivo tumorigenicity assay using indicated cells. Each group contains 5 mice. i Correlation of Gli1 mRNA and circ-SMO in 86 GBM patients and correlation of Gli1 protein and SMO-193a.a. in 86 GBM patients. j Graphic abstract. Encoded by circular SMO (circ-SMO), SMO-193a.a. is required for Shh-induced SMO activation, via interacting with SMO, enhancing SMO cholesterol modification and releasing SMO from the inhibition of patched transmembrane receptors 1 (PTCH1). Moreover, circ-SMO/SMO-193a.a. is positively regulated by FUS, a direct transcriptional target of Gli1. Shh/Gli1/FUS/SMO-193a.a. form a positive feedback loop to sustain HH signaling activation in GBM. Lines show the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. Data are representative from at least three experiments with similar results