Core-binding factor fusion downregulation of ADAR2 RNA editing contributes to AML leukemogenesis.

Adenosine-to-inosine RNA editing, which is catalyzed by adenosine deaminases acting on RNA (ADAR) family of enzymes, ADAR1 and ADAR2, has been shown to contribute to multiple cancers. However, other than the chronic myeloid leukemia blast crisis, relatively little is known about its role in other types of hematological malignancies. Here, we found that ADAR2, but not ADAR1 and ADAR3, was specifically downregulated in the core-binding factor (CBF) acute myeloid leukemia (AML) with t(8;21) or inv(16) translocations. In t(8;21) AML, RUNX1-driven transcription of ADAR2 was repressed by the RUNX1-ETO additional exon 9a fusion protein in a dominant-negative manner. Further functional studies confirmed that ADAR2 could suppress leukemogenesis specifically in t(8;21) and inv16 AML cells dependent on its RNA editing capability. Expression of 2 exemplary ADAR2-regulated RNA editing targets coatomer subunit α and component of oligomeric Golgi complex 3 inhibits the clonogenic growth of human t(8;21) AML cells. Our findings support a hitherto, unappreciated mechanism leading to ADAR2 dysregulation in CBF AML and highlight the functional relevance of loss of ADAR2-mediated RNA editing to CBF AML.

Targeting RNA editing of antizyme inhibitor 1: A potential oligonucleotide-based antisense therapy for cancer.

Dysregulated adenosine-to-inosine (A-to-I) RNA editing is implicated in various cancers. However, no available RNA editing inhibitors have so far been developed to inhibit cancer-associated RNA editing events. Here, we decipher the RNA secondary structure of antizyme inhibitor 1 (AZIN1), one of the best-studied A-to-I editing targets in cancer, by locating its editing site complementary sequence (ECS) at the 3' end of exon 12. Chemically modified antisense oligonucleotides (ASOs) that target the editing region of AZIN1 caused a substantial exon 11 skipping, whereas ECS-targeting ASOs effectively abolished AZIN1 editing without affecting splicing and translation. We demonstrate that complete 2'-O-methyl (2'-O-Me) sugar ring modification in combination with partial phosphorothioate (PS) backbone modification may be an optimal chemistry for editing inhibition. ASO3.2, which targets the ECS, specifically inhibits cancer cell viability in vitro and tumor incidence and growth in xenograft models. Our results demonstrate that this AZIN1-targeting, ASO-based therapeutics may be applicable to a wide range of tumor types.

RNA editing mediates the functional switch of COPA in a novel mechanism of hepatocarcinogenesis.

BACKGROUND & AIMS: RNA editing introduces nucleotide changes in RNA sequences. Recent studies have reported that aberrant adenosine-to-inosine RNA editing is implicated in cancers. Until now, very few functionally important protein-recoding editing targets have been discovered. Here, we investigated the role of a recently discovered protein-recoding editing target COPA (coatomer subunit α) in hepatocellular carcinoma (HCC). METHODS: Clinical implication of COPA editing was studied in a cohort of 125 HCC patients. CRISPR/Cas9-mediated knockout of the editing site complementary sequence (ECS) was used to delete edited COPA transcripts endogenously. COPA editing-mediated change in its transcript or protein stability was investigated upon actinomycin D or cycloheximide treatment, respectively. Functional difference in tumourigenesis between wild-type and edited COPA (COPAWTvs. COPAI164V) and the exact mechanisms were also studied in cell models and mice. RESULTS: ADAR2 binds to double-stranded RNA formed between edited exon 6 and the ECS at intron 6 of COPA pre-mRNA, causing an isoleucine-to-valine substitution at residue 164. Reduced editing of COPA is implicated in the pathogenesis of HCC, and more importantly, it may be involved in many cancer types. Upon editing, COPAWT switches from a tumour-promoting gene to a tumour suppressor that has a dominant-negative effect. Moreover, COPAI164V may undergo protein conformational change and therefore become less stable than COPAWT. Mechanistically, COPAI164V may deactivate the PI3K/AKT/mTOR pathway through downregulation of caveolin-1 (CAV1). CONCLUSIONS: We uncover an RNA editing-associated mechanism of hepatocarcinogenesis by which downregulation of ADAR2 caused the loss of tumour suppressive COPAI164V and concurrent accumulation of tumour-promoting COPAWT in tumours; a rapid degradation of COPAI164V protein and hyper-activation of the PI3K/AKT/mTOR pathway further promote tumourigenesis. LAY SUMMARY: RNA editing is a process in which RNA is changed after it is made from DNA, resulting in an altered gene product. In this study, we found that RNA editing of a gene known as coatomer subunit α (COPA) is lower in tumour samples and discovered that this editing process changes COPA protein from a tumour-promoting form to a tumour-suppressive form. Loss of the edited COPA promotes the development of liver cancer.

Targeting cancer addiction for SALL4 by shifting its transcriptome with a pharmacologic peptide.

Sal-like 4 (SALL4) is a nuclear factor central to the maintenance of stem cell pluripotency and is a key component in hepatocellular carcinoma, a malignancy with no effective treatment. In cancer cells, SALL4 associates with nucleosome remodeling deacetylase (NuRD) to silence tumor-suppressor genes, such as PTEN. Here, we determined the crystal structure of an amino-terminal peptide of SALL4(1-12) complexed to RBBp4, the chaperone subunit of NuRD, at 2.7 Å, and subsequent design of a potent therapeutic SALL4 peptide (FFW) capable of antagonizing the SALL4-NURD interaction using systematic truncation and amino acid substitution studies. FFW peptide disruption of the SALL4-NuRD complex resulted in unidirectional up-regulation of transcripts, turning SALL4 from a dual transcription repressor-activator mode to singular transcription activator mode. We demonstrate that FFW has a target affinity of 23 nM, and displays significant antitumor effects, inhibiting tumor growth by 85% in xenograft mouse models. Using transcriptome and survival analysis, we discovered that the peptide inhibits the transcription-repressor function of SALL4 and causes massive up-regulation of transcripts that are beneficial to patient survival. This study supports the SALL4-NuRD complex as a drug target and FFW as a viable drug candidate, showcasing an effective strategy to accurately target oncogenes previously considered undruggable.

An RNA editing/dsRNA binding-independent gene regulatory mechanism of ADARs and its clinical implication in cancer.

Adenosine-to-inosine (A-to-I) RNA editing, catalyzed by Adenosine DeAminases acting on double-stranded RNA(dsRNA) (ADAR), occurs predominantly in the 3' untranslated regions (3'UTRs) of spliced mRNA. Here we uncover an unanticipated link between ADARs (ADAR1 and ADAR2) and the expression of target genes undergoing extensive 3'UTR editing. Using METTL7A (Methyltransferase Like 7A), a novel tumor suppressor gene with multiple editing sites at its 3'UTR, we demonstrate that its expression could be repressed by ADARs beyond their RNA editing and double-stranded RNA (dsRNA) binding functions. ADARs interact with Dicer to augment the processing of pre-miR-27a to mature miR-27a. Consequently, mature miR-27a targets the METTL7A 3'UTR to repress its expression level. In sum, our study unveils that the extensive 3'UTR editing of METTL7A is merely a footprint of ADAR binding, and there are a subset of target genes that are equivalently regulated by ADAR1 and ADAR2 through their non-canonical RNA editing and dsRNA binding-independent functions, albeit maybe less common. The functional significance of ADARs is much more diverse than previously appreciated and this gene regulatory function of ADARs is most likely to be of high biological importance beyond the best-studied editing function. This non-editing side of ADARs opens another door to target cancer.

ADAR-Mediated RNA Editing Predicts Progression and Prognosis of Gastric Cancer.

BACKGROUD & AIMS: Gastric cancer (GC) is the third leading cause of global cancer mortality. Adenosine-to-inosine RNA editing is a recently described novel epigenetic mechanism involving sequence alterations at the RNA but not DNA level, primarily mediated by ADAR (adenosine deaminase that act on RNA) enzymes. Emerging evidence suggests a role for RNA editing and ADARs in cancer, however, the relationship between RNA editing and GC development and progression remains unknown. METHODS: In this study, we leveraged on the next-generation sequencing transcriptomics to demarcate the GC RNA editing landscape and the role of ADARs in this deadly malignancy. RESULTS: Relative to normal gastric tissues, almost all GCs displayed a clear RNA misediting phenotype with ADAR1/2 dysregulation arising from the genomic gain and loss of the ADAR1 and ADAR2 gene in primary GCs, respectively. Clinically, patients with GCs exhibiting ADAR1/2 imbalance demonstrated extremely poor prognoses in multiple independent cohorts. Functionally, we demonstrate in vitro and in vivo that ADAR-mediated RNA misediting is closely associated with GC pathogenesis, with ADAR1 and ADAR2 playing reciprocal oncogenic and tumor suppressive roles through their catalytic deaminase domains, respectively. Using an exemplary target gene PODXL (podocalyxin-like), we demonstrate that the ADAR2-regulated recoding editing at codon 241 (His to Arg) confers a loss-of-function phenotype that neutralizes the tumorigenic ability of the unedited PODXL. CONCLUSIONS: Our study highlights a major role for RNA editing in GC disease and progression, an observation potentially missed by previous next-generation sequencing analyses of GC focused on DNA alterations alone. Our findings also suggest new GC therapeutic opportunities through ADAR1 enzymatic inhibition or the potential restoration of ADAR2 activity.

CHD1L promotes lineage reversion of hepatocellular carcinoma through opening chromatin for key developmental transcription factors.

UNLABELLED: High-grade tumors with poor differentiation usually show phenotypic resemblance to their developmental ancestral cells. Cancer cells that gain lineage precursor cell properties usually hijack developmental signaling pathways to promote tumor malignant progression. However, the molecular mechanisms underlying this process remain unclear. In this study, the chromatin remodeler chromodomain-helicase-DNA-binding-protein 1-like (CHD1L) was found closely associated with liver development and hepatocellular carcinoma (HCC) tumor differentiation. Expression of CHD1L decreased during hepatocyte maturation and increased progressively from well-differentiated HCCs to poorly differentiated HCCs. Chromatin immunoprecipitation followed by high-throughput deep sequencing found that CHD1L could bind to the genomic sequences of genes related to development. Bioinformatics-aided network analysis indicated that CHD1L-binding targets might form networks associated with developmental transcription factor activation and histone modification. Overexpression of CHD1L conferred ancestral precursor-like properties of HCC cells both in vitro and in vivo. Inhibition of CHD1L reversed tumor differentiation and sensitized HCC cells to sorafenib treatment. Mechanism studies revealed that overexpression of CHD1L could maintain an active "open chromatin" configuration at promoter regions of estrogen-related receptor-beta and transcription factor 4, both of which are important regulators of HCC self-renewal and differentiation. In addition, we found a significant correlation of CHD1L with developmental transcriptional factors and lineage differentiation markers in clinical HCC patients. CONCLUSION: Genomic amplification of chromatin remodeler CHD1L might drive dedifferentiation of HCC toward an ancestral lineage through opening chromatin for key developmental transcriptional factors; further inhibition of CHD1L might "downgrade" poorly differentiated HCCs and provide novel therapeutic strategies.

Allele-specific imbalance of oxidative stress-induced growth inhibitor 1 associates with progression of hepatocellular carcinoma.

BACKGROUND & AIMS: Although there are a few highly penetrant mutations that are linked directly to cancer initiation, more less-penetrant susceptibility alleles have been associated with cancer risk and progression. We used RNA sequence analysis to search for genetic variations associated with pathogenesis of hepatocellular carcinoma (HCC). METHODS: We analyzed 400 paired HCC and adjacent nontumor tissues, along with clinical information, from patients who underwent surgery at Sun Yat-Sen University in Guangzhou, China. Total RNA was extracted from tissues and sequenced, and variations with allele imbalance were identified. Effects of variants on cell functions were investigated in HCC cell lines and tumor xenografts in mice. Variants were associated with patient outcomes. RESULTS: We found a high proportion of allele imbalance in genes related to cellular stress. A nucleotide variation in the Oxidative Stress-Induced Growth Inhibitor 1 (OSGIN1) gene (nt 1494: G-A) resulted in an amino acid substitution (codon 438: Arg-His). The variant form of OSGIN1 was specifically retained in the tumor tissues. Functional assays showed that the common form of OSGIN1 functioned as a tumor suppressor, sensitizing HCC cells to chemotherapeutic agents by inducing apoptosis. However, the variant form of OSGIN1 was less effective. It appeared to affect the translocation of OSGIN1 from the nucleus to mitochondria, which is important for its apoptotic function. The expression pattern and localization of OSGIN1 was altered in HCC specimens, compared with adjacent liver tissue. Levels of OSGIN1 messenger RNA were reduced in 24.7% of HCC specimens, and down-regulation was associated with shorter overall and disease-free survival times of patients. Patients with the OSGIN1 1494A variant had the shortest mean survival time (32.68 mo) among patient subgroups, and their tumor samples had the lowest apoptotic index. CONCLUSIONS: We identified OSGIN1 as a tumor suppressor that is down-regulated or altered in human HCCs. Variants of OSGIN1 detected in HCC samples reduce apoptosis and are associated with shorter survival times of patients.

A disrupted RNA editing balance mediated by ADARs (Adenosine DeAminases that act on RNA) in human hepatocellular carcinoma.

OBJECTIVE: Hepatocellular carcinoma (HCC) is a heterogeneous tumour displaying a complex variety of genetic and epigenetic changes. In human cancers, aberrant post-transcriptional modifications, such as alternative splicing and RNA editing, may lead to tumour specific transcriptome diversity. DESIGN: By utilising large scale transcriptome sequencing of three paired HCC clinical specimens and their adjacent non-tumour (NT) tissue counterparts at depth, we discovered an average of 20 007 inferred A to I (adenosine to inosine) RNA editing events in transcripts. The roles of the double stranded RNA specific ADAR (Adenosine DeAminase that act on RNA) family members (ADARs) and the altered gene specific editing patterns were investigated in clinical specimens, cell models and mice. RESULTS: HCC displays a severely disrupted A to I RNA editing balance. ADAR1 and ADAR2 manipulate the A to I imbalance of HCC via their differential expression in HCC compared with NT liver tissues. Patients with ADAR1 overexpression and ADAR2 downregulation in tumours demonstrated an increased risk of liver cirrhosis and postoperative recurrence and had poor prognoses. Due to the differentially expressed ADAR1 and ADAR2 in tumours, the altered gene specific editing activities, which was reflected by the hyper-editing of FLNB (filamin B, ß) and the hypo-editing of COPA (coatomer protein complex, subunit α), are closely associated with HCC pathogenesis. In vitro and in vivo functional assays prove that ADAR1 functions as an oncogene while ADAR2 has tumour suppressive ability in HCC. CONCLUSIONS: These findings highlight the fact that the differentially expressed ADARs in tumours, which are responsible for an A to I editing imbalance, has great prognostic value and diagnostic potential for HCC.

SPOCK1 is regulated by CHD1L and blocks apoptosis and promotes HCC cell invasiveness and metastasis in mice.

BACKGROUND & AIMS: Chromodomain helicase/adenosine triphosphatase DNA binding protein 1-like (CHD1L) is an SNF2-like transcription factor involved in the development of human hepatocellular carcinoma (HCC). Sparc/osteonectin, cwcv, and kazal-like domains proteoglycan 1 (SPOCK1) is up-regulated by CHD1L; we investigated its role in hepatocellular carcinogenesis. METHODS: We investigated interactions between SPOCK1 and CHD1L using electrophoretic mobility shift and luciferase reporter assays. Levels of SPOCK1 messenger RNA (mRNA) and protein were measured in samples of HCC and adjacent nontumor liver tissues (135 pairs) and compared using Pearson correlation coefficients. Effects of SPOCK1 overexpression and silencing were determined in HCC cell lines (QGY-7703, PLC-8024, BEL-7402, and QGY-7701). RESULTS: The CHD1L protein bound directly to the promoter region (nt-1662 to +34) of SPOCK1 and activated transcription. Levels of SPOCK1 mRNA and protein were increased in 60% of human HCC samples, compared with nontumor live tissues, and was associated significantly with clinical stage. Levels of SPOCK1 mRNA were increased among tumors that became metastatic, compared with those that did not, and among patients with shorter overall and disease-free survival times. Ectopic expression of SPOCK1 in HCC cells increased proliferation, foci formation, and colony formation in soft agar; these cells also formed larger xenograft tumors, more rapidly, in nude mice than control HCC cells. Silencing SPOCK1 expression with short hairpin RNA had the opposite effects. We found that SPOCK1 prevents apoptosis of HCC cells by activating Akt, to block release of cytochrome c and activation of caspase-9 and caspase-3; these effects were reversed with an Akt inhibitor. HCC cells that overexpressed SPOCK1 expressed higher levels of matrix metallopeptidase 9, were more invasive in Matrigel assays, and formed more metastatic nodules in immunodeficient mice than control HCC cells. CONCLUSIONS: CHD1L activates expression of SPOCK1, which activates Akt signaling to block apoptosis and invasion by HCC cells, in culture and in mice. Levels of SPOCK1 increase with progression of human HCC. SPOCK1 might be used as a prognostic factor or therapeutic target.

Serum and glucocorticoid kinase 3 at 8q13.1 promotes cell proliferation and survival in hepatocellular carcinoma.

Amplification of broad regions of 8q is one of the most frequent genetic alterations in hepatocellular carcinoma (HCC), suggesting the existence of oncogenes in addition to MYC at 8q24.21. In this report we examine the potential role of the candidate amplified oncogene serum and glucocorticoid kinase 3 (SGK3) at 8q13.1 in HCC pathogenesis. We found amplification and overexpression of SGK3 was frequently detected in clinical HCC specimens and that SGK3 genomic activation was significantly associated with poor outcome of patients (P = 0.028). Functionally, we found that overexpression of SGK3 in HCC cells increased cell cycle progression through G(1), cell survival, clonogenicity, anchorage-independent growth, and tumor formation in nude mice. In contrast, RNA interference (RNAi) silencing of SGK3 inhibited its oncogenic effects. We provide evidence that SGK3 promotes HCC growth and survival through inactivating glycogen synthase kinase 3 beta and Bcl-2-associated death promoter, respectively. We also found that expression of SGK3, which like AKT is activated by PI3K/PDK1 signaling, has more significance than overexpression of AKT in predicting poor outcome in HCC patients. Taken together, our findings in the present study suggests that the SGK3 pathway may function in parallel with the AKT pathway and undergoes an AKT-independent signaling pathway in the pathogenesis of HCC. Further characterization of SGK3 may provide a prognostic biomarker for HCC outcome prediction and a novel therapeutic target in HCC treatment.

Translationally controlled tumor protein induces mitotic defects and chromosome missegregation in hepatocellular carcinoma development.

UNLABELLED: Emerging evidence implicates the chromodomain helicase/ATPase DNA binding protein 1-like gene (CHD1L) as a specific oncogene in human hepatocellular carcinoma (HCC). To better understand the molecular mechanisms underlying HCC cases carrying CHD1L amplification (>50% HCCs), we identified a CHD1L target, translationally controlled tumor protein (TCTP), and investigated its role in HCC progression. Here, we report that CHD1L protein directly binds to the promoter region (nt -733 to -1,027) of TCTP and activates TCTP transcription. Overexpression of TCTP was detected in 40.7% of human HCC samples analyzed and positively correlated with CHD1L overexpression. Clinically, overexpression of TCTP was significantly associated with the advanced tumor stage (P = 0.037) and overall survival time of HCC patients (P = 0.034). In multivariate analyses, TCTP was determined to be an independent marker associated with poor prognostic outcomes. In vitro and in vivo functional studies in mice showed that TCTP has tumorigenic abilities, and overexpression of TCTP induced by CHD1L contributed to the mitotic defects of tumor cells. Further mechanistic studies demonstrated that TCTP promoted the ubiquitin-proteasome degradation of Cdc25C during mitotic progression, which caused the failure in the dephosphorylation of Cdk1 on Tyr15 and decreased Cdk1 activity. As a consequence, the sudden drop of Cdk1 activity in mitosis induced a faster mitotic exit and chromosome missegregation, which led to chromosomal instability. The depletion experiment proved that the tumorigenicity of TCTP was linked to its role in mitotic defects. CONCLUSION: Collectively, we reveal a novel molecular pathway (CHD1L/TCTP/Cdc25C/Cdk1), which causes the malignant transformation of hepatocytes with the phenotypes of accelerated mitotic progression and the production of aneuploidy.

MicroRNA-375 inhibits tumour growth and metastasis in oesophageal squamous cell carcinoma through repressing insulin-like growth factor 1 receptor.

BACKGROUND: To understand the involvement of micro-RNA (miRNA) in the development and progression of oesophageal squamous cell carcinoma (ESCC), miRNA profiles were compared between tumour and corresponding non-tumour tissues. METHODS: miRCURY LNA array was used to generate miRNA expressing profile. Real-time quantitative PCR was applied to detectthe expression of miR-375 in ESCC samples and its correlation with insulin-like growth factor 1 receptor (IGF1R). Methylation-specific PCR was used to study the methylation status in the promoter region of miR-375. The tumour-suppressive effect of miR-375 was determined by both in-vitro and in-vivo assays. RESULTS: The downregulation of miR-375 was frequently detected in primary ESCC, which was significantly correlated with advanced stage (p=0.003), distant metastasis (p<0.0001), poor overall survival (p=0.048) and disease-free survival (p=0.0006). Promoter methylation of miR-375 was detected in 26 of 45 (57.8%) ESCC specimens. Functional assays demonstrated that miR-375 could inhibit clonogenicity, cell motility, cell proliferation, tumour formation and metastasis in mice. Further study showed that miR-375 could interact with the 3'-untranslated region of IGF1R and downregulate its expression. In clinical specimens, the expression of IGF1R was also negatively correlated with miR-375 expression (p=0.008). CONCLUSIONS: This study demonstrates that miR-375 has a strong tumour-suppressive effect through inhibiting the expression of IGF1R. The downregulation of miR-375, which is mainly caused by promoter methylation, is one of the molecular mechanisms involved in the development and progression of ESCC.

Towards a Rapid-Turnaround Low-Depth Unbiased Metagenomics Sequencing Workflow on the Illumina Platforms.

Unbiased metagenomic sequencing is conceptually well-suited for first-line diagnosis as all known and unknown infectious entities can be detected, but costs, turnaround time and human background reads in complex biofluids, such as plasma, hinder widespread deployment. Separate preparations of DNA and RNA also increases costs. In this study, we developed a rapid unbiased metagenomics next-generation sequencing (mNGS) workflow with a human background depletion method (HostEL) and a combined DNA/RNA library preparation kit (AmpRE) to address this issue. We enriched and detected bacterial and fungal standards spiked in plasma at physiological levels with low-depth sequencing (<1 million reads) for analytical validation. Clinical validation also showed 93% of plasma samples agreed with the clinical diagnostic test results when the diagnostic qPCR had a Ct < 33. The effect of different sequencing times was evaluated with the 19 h iSeq 100 paired end run, a more clinically palatable simulated iSeq 100 truncated run and the rapid 7 h MiniSeq platform. Our results demonstrate the ability to detect both DNA and RNA pathogens with low-depth sequencing and that iSeq 100 and MiniSeq platforms are compatible with unbiased low-depth metagenomics identification with the HostEL and AmpRE workflow.

SCYL1 binding protein 1 promotes the ubiquitin-dependent degradation of Pirh2 and has tumor-suppressive function in the development of hepatocellular carcinoma.

Pirh2 is a Ring-H2 domain containing E3 ubiquitin ligase that targets several important tumor suppressor genes for proteasomal degradation. Overexpression of Pirh2 is frequently detected in many clinical tumor tissues including hepatocellular carcinoma (HCC). However, the molecular mechanism of Pirh2 activation in tumorigenesis still remains poorly understood. In this study, we find a Pirh2-binding protein, SCYL1 binding protein 1 (SCYL1BP1), that can promote the ubiquitin-dependent degradation of Pirh2. SCYL1BP1 colocalized with Pirh2 in the cytoplasm and prevented its localization to the nucleus. Ectopic expression of SCYL1BP1 increased the expression of p53 and further inhibited the G(1)/S transition of HCC cell lines. Conversely, knock down of SCYL1BP1 restored the expression of Pirh2 and inhibited p53 at protein level. Functional assays found that reintroduction of SCYL1BP1 into HCC cell lines significantly inhibited cell proliferation, foci formation, colony formation in soft agar and tumor formation in nude mice, suggesting the strong tumor-suppressive function of SCYL1BP1 in HCC progression. Furthermore, SCYL1BP1 was found to be frequently downregulated in HCC clinical specimens compared to their paired non-tumor tissues by immunohistochemical staining. Taken together, our data suggested that the interaction of SCYL1BP1/Pirh2 could accelerate Pirh2 degradation through an ubiquitin-dependent pathway. SCYL1BP1 may function as an important tumor suppressor gene in HCC development.

Clinical significance of CHD1L in hepatocellular carcinoma and therapeutic potentials of virus-mediated CHD1L depletion.

BACKGROUND: Hepatocellular carcinoma (HCC) is among the most lethal of human malignancies. It is difficult to detect early, has a high recurrence rate and is refractory to chemotherapies. Amplification of 1q21 is one of the most frequent genetic alterations in HCC. CHD1L is a newly identified oncogene responsible for 1q21 amplification. This study aims to investigate the role of CHD1L in predicting prognosis and chemotherapy response of patients with HCC, its chemoresistant mechanism and whether virus-mediated CHD1L silencing has therapeutic potentials for HCC treatment. METHODS: The clinical significance of CHD1L in a cohort of 109 HCC cases including 50 cases who received transarterial chemoembolisation treatment was assessed by clinical correlation and Kaplan-Meier analyses. A CHD1L-overexpressing cell model was generated and the mechanism of chemoresistance involving CHD1L was investigated. An adenovirus-mediated silencing method was used to knockdown CHD1L, and its effects on tumorigenicity and chemoresistance were investigated in vivo and in vitro. RESULTS: Overexpression of CHD1L was significantly associated with tumour microsatellite formation (p = 0.045), advanced tumour stage (p = 0.018), overall survival time (p = 0.002), overall survival time of patients who received transarterial chemoembolisation treatment (p = 0.028) and chemoresistance (p = 0.020) in HCC. Interestingly, CHD1L could inhibit apoptosis induced by 5-fluorourail (5-FU) but not doxorubicin. The mechanistic study revealed that the involvement of the Nur77-mediated pathway in chemotherapeutic agent-induced apoptosis can dictate if CHD1L could confer resistance to chemotherapy. Furthermore, an adenoviral vector containing short hairpin RNAs against CHD1L (CHD1L-shRNAs) could suppress cell growth, clonogenicity and chemoresistance to 5-FU. An in vivo study found that CHD1L-shRNAs could inhibit xenograft tumour growth and increase the sensitivity of tumour cells to 5-FU in nude mice. CONCLUSIONS: This study highlighted for the first time the prognostic value of CHD1L in HCC and the potential application of virus-mediated CHD1L silencing in HCC treatment.

HNRNPM controls circRNA biogenesis and splicing fidelity to sustain cancer cell fitness.

High spliceosome activity is a dependency for cancer cells, making them more vulnerable to perturbation of the splicing machinery compared to normal cells. To identify splicing factors important for prostate cancer (PCa) fitness, we performed pooled shRNA screens in vitro and in vivo. Our screens identified heterogeneous nuclear ribonucleoprotein M (HNRNPM) as a regulator of PCa cell growth. RNA- and eCLIP-sequencing identified HNRNPM binding to transcripts of key homeostatic genes. HNRNPM binding to its targets prevents aberrant exon inclusion and backsplicing events. In both linear and circular mis-spliced transcripts, HNRNPM preferentially binds to GU-rich elements in long flanking proximal introns. Mimicry of HNRNPM-dependent linear-splicing events using splice-switching-antisense-oligonucleotides was sufficient to inhibit PCa cell growth. This suggests that PCa dependence on HNRNPM is likely a result of mis-splicing of key homeostatic coding and non-coding genes. Our results have further been confirmed in other solid tumors. Taken together, our data reveal a role for HNRNPM in supporting cancer cell fitness. Inhibition of HNRNPM activity is therefore a potential therapeutic strategy in suppressing growth of PCa and other solid tumors.

Chromodomain helicase/adenosine triphosphatase DNA binding protein 1-like (CHD1l) gene suppresses the nucleus-to-mitochondria translocation of nur77 to sustain hepatocellular carcinoma cell survival.

UNLABELLED: Amplification of 1q21 has been detected in 58% to 78% of primary hepatocellular carcinoma cases, suggesting that one or more oncogenes within the amplicon play a critical role in the development of this disease. The chromodomain helicase/adenosine triphosphatase DNA binding protein 1-like gene (CHD1L) is a recently identified oncogene localized at 1q21. Our previous studies have demonstrated that CHD1L has strong tumorigenic ability and confers high susceptibility to spontaneous tumors in a CHD1L-transgenic mouse model. In this study, we demonstrate that the antiapoptotic ability of CHD1L is associated with its interaction with Nur77, a critical member of a p53-independent apoptotic pathway. As the first cellular protein identified to bind Nur77, CHD1L is able to inhibit the nucleus-to-mitochondria translocation of Nur77, which is the key step of Nur77-mediated apoptosis, resulting in the hindrance of the release of cytochrome c and the initiation of apoptosis. Knock-down of CHD1L expression by RNA interference could rescue the mitochondrial targeting of Nur77 and the subsequent apoptosis. Further studies found that the C-terminal Macro domain of CHD1L is responsible for the interaction with Nur77, and a CHD1L mutant lacking residues 600-897 failed to interact with Nur77 and prevented Nur77-mediated apoptosis. More importantly, we found that the inhibition of Nur77-mediated apoptosis by endogenous CHD1L is a critical biological cellular process in hepatocarcinogenesis. CONCLUSION: We demonstrate in this study that overexpression of CHD1L could sustain tumor cell survival by preventing Nur77-mediated apoptosis.

Chromosome 1q21 amplification and oncogenes in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is among the most lethal of human malignancies. During human multistep hepatocarcinogenesis, genomic gain represents an important mechanism in the activation of proto-oncogenes. In many circumstances, activated oncogenes hold clinical implications both as prognostic markers and targets for cancer therapeutics. Gain of chromosome 1q copy is one of the most frequently detected alterations in HCC and 1q21 is the most frequent minimal amplifying region (MAR). A better understanding of the physiological and pathophysiological roles of target genes within 1q21 amplicon will significantly improve our knowledge in HCC pathogenesis, and may lead to a much more effective management of HCC bearing amplification of 1q21. Such knowledge has long term implications for the development of new therapeutic strategies for HCC treatment. Our research group and others, focused on the identification and characterization of 1q21 target genes such as JTB, CKS1B, and CHD1L in HCC progression. In this review, we will summarize the current scientific knowledge of known target genes within 1q21 amplicon and the precise oncogenic mechanisms of CHD1L will be discussed in detail.

Suppression of adenosine-to-inosine (A-to-I) RNA editome by death associated protein 3 (DAP3) promotes cancer progression.

RNA editing introduces nucleotide changes in RNA sequences. Recent studies have reported that aberrant A-to-I RNA editing profiles are implicated in cancers. Albeit changes in expression and activity of ADAR genes are thought to have been responsible for the dysregulated RNA editome in diseases, they are not always correlated, indicating the involvement of secondary regulators. Here, we uncover DAP3 as a potent repressor of editing and a strong oncogene in cancer. DAP3 mainly interacts with the deaminase domain of ADAR2 and represses editing via disrupting association of ADAR2 with its target transcripts. PDZD7, an exemplary DAP3-repressed editing target, undergoes a protein recoding editing at stop codon [Stop →Trp (W)]. Because of editing suppression by DAP3, the unedited PDZD7WT, which is more tumorigenic than edited PDZD7Stop518W, is accumulated in tumors. In sum, cancer cells may acquire malignant properties for their survival advantage through suppressing RNA editome by DAP3.