Circular RNAs (circRNAs) are recognized as functional non-coding transcripts in eukaryotic cells. Recent evidence has indicated that even though circRNAs are generally expressed at low levels, they ...may be involved in many physiological or pathological processes, such as gene regulation, tissue development and carcinogenesis. Although the 'microRNA sponge' function is well characterized, most circRNAs do not contain perfect trapping sites for microRNAs, which suggests the possibility that circRNAs have functions that have not yet been defined. In this study, we show that a circRNA containing an open reading frame (ORF) driven by the internal ribosome entry site (IRES) can translate a functional protein. The circular form of the SNF2 histone linker PHD RING helicase (SHPRH) gene encodes a novel protein that we termed SHPRH-146aa. Circular SHPRH (circ-SHPRH) uses overlapping genetic codes to generate a 'UGA' stop codon, which results in the translation of the 17 kDa SHPRH-146aa. Both circ-SHPRH and SHPRH-146aa are abundantly expressed in normal human brains and are down-regulated in glioblastoma. The overexpression of SHPRH-146aa in U251 and U373 glioblastoma cells reduces their malignant behavior and tumorigenicity in vitro and in vivo. Mechanistically, SHPRH-146aa protects full-length SHPRH from degradation by the ubiquitin proteasome. Stabilized SHPRH sequentially ubiquitinates proliferating cell nuclear antigen (PCNA) as an E3 ligase, leading to inhibited cell proliferation and tumorigenicity. Our findings provide a novel perspective regarding circRNA function in physiological and pathological processes. Specifically, SHPRH-146aa generated from overlapping genetic codes of circ-SHPRH is a tumor suppressor in human glioblastoma.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Circular RNAs (circRNAs) are a large class of transcripts in the mammalian genome. Although the translation of circRNAs was reported, additional coding circRNAs and the functions of their translated ...products remain elusive. Here, we demonstrate that an endogenous circRNA generated from a long noncoding RNA encodes regulatory peptides. Through ribosome nascent-chain complex-bound RNA sequencing (RNC-seq), we discover several peptides potentially encoded by circRNAs. We identify an 87-amino-acid peptide encoded by the circular form of the long intergenic non-protein-coding RNA p53-induced transcript (LINC-PINT) that suppresses glioblastoma cell proliferation in vitro and in vivo. This peptide directly interacts with polymerase associated factor complex (PAF1c) and inhibits the transcriptional elongation of multiple oncogenes. The expression of this peptide and its corresponding circRNA are decreased in glioblastoma compared with the levels in normal tissues. Our results establish the existence of peptides encoded by circRNAs and demonstrate their potential functions in glioblastoma tumorigenesis.
Activated EGFR signalling drives tumorigenicity in 50% of glioblastoma (GBM). However, EGFR-targeting therapy has proven ineffective in treating patients with GBM, indicating that there is redundant ...EGFR activation. Circular RNAs are covalently closed RNA transcripts that are involved in various physiological and pathological processes. Herein, we report an additional activation mechanism of EGFR signalling in GBM by an undescribed secretory E-cadherin protein variant (C-E-Cad) encoded by a circular E-cadherin (circ-E-Cad) RNA through multiple-round open reading frame translation. C-E-Cad is overexpressed in GBM and promotes glioma stem cell tumorigenicity. C-E-Cad activates EGFR independent of EGF through association with the EGFR CR2 domain using a unique 14-amino-acid carboxy terminus, thereby maintaining glioma stem cell tumorigenicity. Notably, inhibition of C-E-Cad markedly enhances the antitumour activity of therapeutic anti-EGFR strategies in GBM. Our results uncover a critical role of C-E-Cad in stimulating EGFR signalling and provide a promising approach for treating EGFR-driven GBM.
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
The RTK/PI3K/AKT pathway plays key roles in the development and progression of many cancers, including GBM. As a regulatory molecule and a potential drug target, the oncogenic role of AKT has been ...substantially studied. Three isoforms of AKT have been identified, including AKT1, AKT2 and AKT3, but their individual functions in GBM remain controversial. Moreover, it is not known if there are more AKT alternative splicing variants.
High-throughput RNA sequencing and quantitative reverse transcription-PCR were used to identify the differentially expressed circRNAs in GBM samples and in paired normal tissues. High throughput RNA sequencing was used to identify circ-AKT3 regulated signaling pathways. Mass spectrometry, western blotting and immunofluorescence staining analyses were used to validate AKT3-174aa expression. The tumor suppressive role of AKT3-174aa was validated in vitro and in vivo. The competing interaction between AKT3-174aa and p-PDK1 was investigated by mass spectrometry and immunoprecipitation analyses.
Circ-AKT3 is a previously uncharacterized AKT transcript variant. Circ-AKT3 is expressed at low levels in GBM tissues compared with the expression in paired adjacent normal brain tissues. Circ-AKT3 encodes a 174 amino acid (aa) novel protein, which we named AKT3-174aa, by utilizing overlapping start-stop codons. AKT3-174aa overexpression decreased the cell proliferation, radiation resistance and in vivo tumorigenicity of GBM cells, while the knockdown of circ-AKT3 enhanced the malignant phenotypes of astrocytoma cells. AKT3-174aa competitively interacts with phosphorylated PDK1, reduces AKT-thr308 phosphorylation, and plays a negative regulatory role in modulating the PI3K/AKT signal intensity.
Our data indicate that the impaired circRNA expression of the AKT3 gene contributes to GBM tumorigenesis, and our data corroborate the hypothesis that restoring AKT3-174aa while inhibiting activated AKT may provide more benefits for certain GBM patients.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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.
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.
Abstract
Background
Triple negative breast cancer (TNBC) remains the most challenging breast cancer subtype so far. Specific therapeutic approaches have rarely achieved clinical improvements in ...treatment of TNBC patients and effective molecular biomarkers are largely unknown.
Methods
We used paired TNBC samples and high throughput RNA sequencing to identify differentially expressed circRNAs. Sucrose gradient polysome fractionation assay, antibody and Mass spectra were used to validate active circRNA translation. The novel protein function was validated in vitro and in vivo by gain or loss of function assays. Mechanistic results were concluded by immunoprecipitation analyses and kinase activity assay.
Results
Circular HER2 RNA (circ-HER2) encoded a novel protein, HER2–103. Unexpectedly, while HER2 mRNA and protein were barely detected, circ-HER2/HER2–103 was expressed in ~ 30% TNBC clinical samples. Circ-HER2/HER2–103 positive TNBC patients harbored worse overall prognosis than circ-HER2/HER2–103 negative patients. Knockdown circ-HER2 inhibited TNBC cells proliferation, invasion and tumorigenesis in vitro and in vivo, suggesting the critical role of circ-HER2/HER2–103 in TNBC tumorigenicity. Mechanistically, HER2–103 promoted homo/hetero dimerization of epidermal growth factor receptor (EGFR)/HER3, sustained AKT phosphorylation and downstream malignant phenotypes. Furthermore, HER2–103 shared most of the same amino acid sequences as HER2 CR1 domain which could be antagonized by Pertuzumab, a clinical used HER2 antibody. Pertuzumab markedly attenuated in vivo tumorigenicity of circ-HER2/HER2–103 expressing TNBC cells but showed no effects in circ-HER2/HER2–103 negative TNBC cells.
Conclusion
Our results not only demonstrated that certain TNBCs were not truly ‘HER2 negative’ but also highlighted the clinical implications of Pertuzumab in circ-HER2/HER2–103 expressing TNBC patients.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Glioblastoma (GBM) is a highly aggressive primary brain tumour and is resistant to nearly all available treatments, including natural killer (NK) cell immunotherapy. However, the factors ...mediating NK cell evasion in GBM remain largely unclear. Here, we report that EZH2-92aa, a protein encoded by circular EZH2, is overexpressed in GBM and induces the immune evasion of GBM stem cells (GSCs) from NK cells. Positively regulated by DEAD-box helicase 3 (DDX3), EZH2-92aa directly binds the major histocompatibility complex class I polypeptide-related sequence A/B (MICA/B) promoters and represses their transcription; it also indirectly represses UL16-binding protein (ULBP) transcription by stabilizing EZH2. The downregulation of NK group 2D ligands (NKG2DLs, including MICA/B and ULBPs) in GSCs mediates NK cell resistance. Moreover, stable EZH2-92aa knockdown enhances NK cell-mediated GSC eradication in vitro and in vivo and synergizes with anti-PD1 therapy. Our results highlight the immunosuppressive function of EZH2-92aa in inhibiting the NK cell response in GBM and the clinical potential of targeting EZH2-92aa for NK-cell-directed immune therapy.
Activated by its single ligand, hepatocyte growth factor (HGF), the receptor tyrosine kinase MET is pivotal in promoting glioblastoma (GBM) stem cell self-renewal, invasiveness and tumorigenicity. ...Nevertheless, HGF/MET-targeted therapy has shown limited clinical benefits in GBM patients, suggesting hidden mechanisms of MET signalling in GBM. Here, we show that circular MET RNA (circMET) encodes a 404-amino-acid MET variant (MET404) facilitated by the N
-methyladenosine (m
A) reader YTHDF2. Genetic ablation of circMET inhibits MET404 expression in mice and attenuates MET signalling. Conversely, MET404 knock-in (KI) plus P53 knock-out (KO) in mouse astrocytes initiates GBM tumorigenesis and shortens the overall survival. MET404 directly interacts with the MET β subunit and forms a constitutively activated MET receptor whose activity does not require HGF stimulation. High MET404 expression predicts poor prognosis in GBM patients, indicating its clinical relevance. Targeting MET404 through a neutralizing antibody or genetic ablation reduces GBM tumorigenicity in vitro and in vivo, and combinatorial benefits are obtained with the addition of a traditional MET inhibitor. Overall, we identify a MET variant that promotes GBM tumorigenicity, offering a potential therapeutic strategy for GBM patients, especially those with MET hyperactivation.
Mol Cancer 18, 131 (2019) https://doi.org/10.1186/s12943-019-1056-5 Following publication of the original article 1, errors were identified in the images presented in Figs. 3 and 4; specifically: * ...Fig. 3C Representative images showing plate colony formations from different cell lines were incorrectly selected during figure assembly; this affects circ-AKT3 IRES mut U373 (bottom left in left-hand colony formations), and circ-AKT3 shRNA control SW1783 and HS683 (top and bottom left in right-hand colony formations). The correct representative images are now used * Fig. 4G Representative images of the control group for Empty Vector U373 (bottom left cell line) was incorrectly used. RAW_REF_TEXT Correction /RAW_REF_TEXT RAW_REF_TEXT Open Access /RAW_REF_TEXT RAW_REF_TEXT Published:07 June 2022 /RAW_REF_TEXT Correction: A novel tumor suppressor protein encoded by circular AKT3 RNA inhibits glioblastoma tumorigenicity by competing with active phosphoinositide-dependent Kinase-1 RAW_REF_TEXT Xin Xia1,2, /RAW_REF_TEXT RAW_REF_TEXT Xixi Li1,2, /RAW_REF_TEXT RAW_REF_TEXT Fanying Li1,2, /RAW_REF_TEXT RAW_REF_TEXT Xujia Wu1,2, /RAW_REF_TEXT RAW_REF_TEXT Maolei Zhang1,2, /RAW_REF_TEXT RAW_REF_TEXT Huangkai Zhou1,2, /RAW_REF_TEXT RAW_REF_TEXT Nunu Huang1,2, /RAW_REF_TEXT RAW_REF_TEXT Xuesong Yang1,2, /RAW_REF_TEXT RAW_REF_TEXT Feizhe Xiao3, /RAW_REF_TEXT RAW_REF_TEXT Dawei Liu4, /RAW_REF_TEXT RAW_REF_TEXT Lixuan Yang1,2 & /RAW_REF_TEXT RAW_REF_TEXT Nu Zhang 5,6 /RAW_REF_TEXT Molecular Cancer volume 21, Article number: 124 (2022) Cite this article RAW_REF_TEXT 181 Accesses /RAW_REF_TEXT RAW_REF_TEXT Metrics details /RAW_REF_TEXT The Original Article was published on 30 August 2019 Correction to: Mol Cancer 18, 131 (2019) https://doi.org/10.1186/s12943-019-1056-5 Following publication of the original article 1, errors were identified in the images presented in Figs. 3 and 4; specifically: RAW_REF_TEXT Fig. 3C Representative images showing plate colony formations from different cell lines were incorrectly selected during figure assembly; this affects circ-AKT3 IRES mut U373 (bottom left in left-hand colony formations), and circ-AKT3 shRNA control SW1783 and HS683 (top and bottom left in right-hand colony formations). The correct representative images are now used /RAW_REF_TEXT RAW_REF_TEXT Fig. 4G Representative images of the control group for Empty Vector U373 (bottom left cell line) was incorrectly used. Correction Open Access Published:07 June 2022 /RAW_REF_TEXT Correction: A novel tumor suppressor protein encoded by circular AKT3 RNA inhibits glioblastoma tumorigenicity by competing with active phosphoinositide-dependent Kinase-1 RAW_REF_TEXT Xin Xia1,2, Xixi Li1,2, Fanying Li1,2, Xujia Wu1,2, Maolei Zhang1,2, Huangkai Zhou1,2, Nunu Huang1,2, Xuesong Yang1,2, Feizhe Xiao3, Dawei Liu4, Lixuan Yang1,2 & Nu Zhang 5,6 /RAW_REF_TEXT Molecular Cancer volume 21, Article number: 124 (2022) Cite this article RAW_REF_TEXT 181 Accesses Metrics details /RAW_REF_TEXT The Original Article was published on 30 August 2019 Correction to: Mol Cancer 18, 131 (2019) https://doi.org/10.1186/s12943-019-1056-5 Following publication of the original article 1, errors were identified in the images presented in Figs. 3 and 4; specifically: RAW_REF_TEXT Fig. 3C Representative images showing plate colony formations from different cell lines were incorrectly selected during figure assembly; this affects circ-AKT3 IRES mut U373 (bottom left in left-hand colony formations), and circ-AKT3 shRNA control SW1783 and HS683 (top and bottom left in right-hand colony formations). The correct representative images are now used Fig. 4G Representative images of the control group for Empty Vector U373 (bottom left cell line) was incorrectly used.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In the published article 1, an error was noticed in Fig. 6B. The western blot results were reversed between the overexpression group and the knockdown group of circ-AKT3. The corrected and updated ...Fig. 6 is provided below. This error does not affect the findings or conclusions of the article.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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