Pseudouridine synthase 1 promotes hepatocellular carcinoma through mRNA pseudouridylation to enhance the translation of oncogenic mRNAs.

BACKGROUND AND AIMS: Pseudouridine is a prevalent RNA modification and is highly present in the serum and urine of patients with HCC. However, the role of pseudouridylation and its modifiers in HCC remains unknown. We investigated the function and underlying mechanism of pseudouridine synthase 1 (PUS1) in HCC. APPROACH AND RESULTS: By analyzing the TCGA data set, PUS1 was found to be significantly upregulated in human HCC specimens and positively correlated with tumor grade and poor prognosis of HCC. Knockdown of PUS1 inhibited cell proliferation and the growth of tumors in a subcutaneous xenograft mouse model. Accordingly, increased cell proliferation and tumor growth were observed in PUS1-overexpressing cells. Furthermore, overexpression of PUS1 significantly accelerates tumor formation in a mouse HCC model established by hydrodynamic tail vein injection, while knockout of PUS1 decreases it. Additionally, PUS1 catalytic activity is required for HCC tumorigenesis. Mechanistically, we profiled the mRNA targets of PUS1 by utilizing surveying targets by apolipoprotein B mRNA-editing enzyme 1 (APOBEC1)-mediated profiling and found that PUS1 incorporated pseudouridine into mRNAs of a set of oncogenes, thereby endowing them with greater translation capacity. CONCLUSIONS: Our study highlights the critical role of PUS1 and pseudouridylation in HCC development, and provides new insight that PUS1 enhances the protein levels of a set of oncogenes, including insulin receptor substrate 1 (IRS1) and c-MYC, by means of pseudouridylation-mediated mRNA translation.

RNA-binding protein CCDC137 activates AKT signaling and promotes hepatocellular carcinoma through a novel non-canonical role of DGCR8 in mRNA localization.

BACKGROUND: RNA binding proteins (RBPs)-regulated gene expression play a vital role in various pathological processes, including the progression of cancer. However, the role of RBP in hepatocellular carcinoma (HCC) remains much unknown. In this study, we aimed to explore the contribution of RBP CCDC137 in HCC development. METHODS: We analyzed the altered expression level and clinical significance of CCDC137 in database and HCC specimens. In vitro cell assays and in vivo spontaneous mouse models were used to assess the function of CCDC137. Finally, the molecular mechanisms of how CCDC137 regulates gene expression and promotes HCC was explored. RESULTS: CCDC137 is aberrantly upregulated in HCC and correlates with poor clinical outcomes in HCC patients. CCDC137 markedly promoted HCC proliferation and progression in vitro and in vivo. Mechanistically, CCDC137 binds with FOXM1, JTV1, LASP1 and FLOT2 mRNAs, which was revealed by APOBEC1-mediated profiling, to increase their cytoplasmic localization and thus enhance their protein expressions. Upregulation of FOXM1, JTV1, LASP1 and FLOT2 subsequently synergistically activate AKT signaling and promote HCC. Interestingly, we found that CCDC137 binds with the microprocessor protein DGCR8 and DGCR8 has a novel non-canonical function in mRNA subcellular localization, which mediates the cytoplasmic distribution of mRNAs regulated by CCDC137. CONCLUSIONS: Our results identify a critical proliferation-related role of CCDC137 and reveal a novel CCDC137/DGCR8/mRNA localization/AKT axis in HCC progression, which provide a potential target for HCC therapy.

The Conserved LncRNA DIO3OS Restricts Hepatocellular Carcinoma Stemness by Interfering with NONO-Mediated Nuclear Export of ZEB1 mRNA.

Hepatocellular carcinoma (HCC) is an aggressive and fatal disease caused by a subset of cancer stem cells (CSCs). It is estimated that there are approximately 100 000 long noncoding RNAs (lncRNAs) in humans. However, the mechanisms by which lncRNAs affect tumor stemness remain poorly understood. In the present study, it is found that DIO3OS is a conserved lncRNA that is generally downregulated in multiple cancers, including HCC, and its low expression correlates with poor clinical outcomes in HCC. In in vitro cancer cell lines and an in vivo spontaneous HCC mouse model, DIO3OS markedly represses tumor development via its suppressive role in CSCs through downregulation of zinc finger E-box binding homeobox 1 (ZEB1). Interestingly, DIO3OS represses ZEB1 post-transcriptionally without affecting its mRNA levels. Subsequent experiments show that DIO3OS interacts with the NONO protein and restricts NONO-mediated nuclear export of ZEB1 mRNA. Overall, these findings demonstrate that the DIO3OS-NONO-ZEB1 axis restricts HCC development and offers a valuable candidate for CSC-targeted therapeutics for HCC.

mascRNA and its parent lncRNA MALAT1 promote proliferation and metastasis of hepatocellular carcinoma cells by activating ERK/MAPK signaling pathway.

MALAT1-associated small cytoplasmic RNA (mascRNA) is a cytoplasmic tRNA-like small RNA derived from nucleus-located long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). While MALAT1 was extensively studied and was found to function in multiple cellular processes, including tumorigenesis and tumor progression, the role of mascRNA was largely unknown. Here we show that mascRNA is upregulated in multiple cancer cell lines and hepatocellular carcinoma (HCC) clinical samples. Using HCC cells as model, we found that mascRNA and its parent lncRNA MALAT1 can both promote cell proliferation, migration, and invasion in vitro. Correspondingly, both of them can enhance the tumor growth in mice subcutaneous tumor model and can promote metastasis by tail intravenous injection of HCC cells. Furthermore, we revealed that mascRNA and MALAT1 can both activate ERK/MAPK signaling pathway, which regulates metastasis-related genes and may contribute to the aggressive phenotype of HCC cells. Our results indicate a coordination in function and mechanism of mascRNA and MALAT1 during development and progress of HCC, and provide a paradigm for deciphering tRNA-like structures and their parent transcripts in mammalian cells.

The landscape of long noncoding RNA-involved and tumor-specific fusions across various cancers.

The majority of the human genome encodes long noncoding RNA (lncRNA) genes, critical regulators of various cellular processes, which largely outnumber protein-coding genes. However, lncRNA-involved fusions have not been surveyed and characterized yet. Here, we present a systematic study of the lncRNA fusion landscape across cancer types and identify >30 000 high-confidence tumor-specific lncRNA fusions (using 8284 tumor and 6946 normal samples). Fusions positively correlated with DNA damage and cancer stemness and were specifically low in microsatellite instable (MSI)-High or virus-infected tumors. Moreover, fusions distribute differently among cancer molecular subtypes, but with shared enrichment in tumors that are microsatellite stable (MSS), with high somatic copy number alterations (SCNA), and with poor survival. Importantly, we find a potentially new mechanism, mediated by enhancer RNAs (eRNA), which generates secondary fusions that form densely connected fusion networks with many fusion hubs targeted by FDA-approved drugs. Finally, we experimentally validate functions of two tumor-promoting chimeric proteins derived from mRNA-lncRNA fusions, KDM4B-G039927 and EPS15L1-lncOR7C2-1. The EPS15L1 fusion protein may regulate (Gasdermin E) GSDME, critical in pyroptosis and anti-tumor immunity. Our study completes the fusion landscape in cancers, sheds light on fusion mechanisms, and enriches lncRNA functions in tumorigenesis and cancer progression.

RNA sequencing: new technologies and applications in cancer research.

Over the past few decades, RNA sequencing has significantly progressed, becoming a paramount approach for transcriptome profiling. The revolution from bulk RNA sequencing to single-molecular, single-cell and spatial transcriptome approaches has enabled increasingly accurate, individual cell resolution incorporated with spatial information. Cancer, a major malignant and heterogeneous lethal disease, remains an enormous challenge in medical research and clinical treatment. As a vital tool, RNA sequencing has been utilized in many aspects of cancer research and therapy, including biomarker discovery and characterization of cancer heterogeneity and evolution, drug resistance, cancer immune microenvironment and immunotherapy, cancer neoantigens and so on. In this review, the latest studies on RNA sequencing technology and their applications in cancer are summarized, and future challenges and opportunities for RNA sequencing technology in cancer applications are discussed.

Epstein-Barr virus-derived circular RNA LMP2A induces stemness in EBV-associated gastric cancer.

Cancer stem cells (CSCs) are cancer-initiating cells that are not only a source of tumorigenesis but also the cause of tumour progression, metastasis and therapy resistance. EBV-associated gastric cancer (EBVaGC) is a distinct subtype of gastric cancer with unique clinicopathological and molecular features. However, whether CSCs exist in EBVaGC, and the tumorigenic mechanism of EBV, remains unclear. Here, NOD/SCID mice were injected subcutaneously with the EBVaGC cell line SNU719 and treated with 5-fluorouracil weekly. Successive generations of xenografts yielded a highly malignant EBVaGC cell line, SNU-4th, which displays properties of CSCs and mainly consists of CD44+ CD24- cells. In SNU-4th cells, an EBV-encoded circRNA, ebv-circLMP2A, expression increased and plays crucial roles in inducing and maintaining stemness phenotypes through targeting miR-3908/TRIM59/p53 axis. Additionally, high expression of ebv-circLMP2A is significantly associated with metastasis and poor prognosis in patients with EBVaGC. These findings not only provide evidence for the existence of CSCs in EBVaGC and elucidate the pathogenic mechanism of ebv-circLMP2A in EBVaGC, but also provide a promising therapeutic target for EBVaGC.

BAP1 links metabolic regulation of ferroptosis to tumour suppression.

The roles and regulatory mechanisms of ferroptosis (a non-apoptotic form of cell death) in cancer remain unclear. The tumour suppressor BRCA1-associated protein 1 (BAP1) encodes a nuclear deubiquitinating enzyme to reduce histone 2A ubiquitination (H2Aub) on chromatin. Here, integrated transcriptomic, epigenomic and cancer genomic analyses link BAP1 to metabolism-related biological processes, and identify cystine transporter SLC7A11 as a key BAP1 target gene in human cancers. Functional studies reveal that BAP1 decreases H2Aub occupancy on the SLC7A11 promoter and represses SLC7A11 expression in a deubiquitinating-dependent manner, and that BAP1 inhibits cystine uptake by repressing SLC7A11 expression, leading to elevated lipid peroxidation and ferroptosis. Furthermore, we show that BAP1 inhibits tumour development partly through SLC7A11 and ferroptosis, and that cancer-associated BAP1 mutants lose their abilities to repress SLC7A11 and to promote ferroptosis. Together, our results uncover a previously unappreciated epigenetic mechanism coupling ferroptosis to tumour suppression.

Energy stress-induced lncRNA FILNC1 represses c-Myc-mediated energy metabolism and inhibits renal tumor development.

The roles of long non-coding RNAs in cancer metabolism remain largely unexplored. Here we identify FILNC1 (FoxO-induced long non-coding RNA 1) as an energy stress-induced long non-coding RNA by FoxO transcription factors. FILNC1 deficiency in renal cancer cells alleviates energy stress-induced apoptosis and markedly promotes renal tumor development. We show that FILNC1 deficiency leads to enhanced glucose uptake and lactate production through upregulation of c-Myc. Upon energy stress, FILNC1 interacts with AUF1, a c-Myc mRNA-binding protein, and sequesters AUF1 from binding c-Myc mRNA, leading to downregulation of c-Myc protein. FILNC1 is specifically expressed in kidney, and is downregulated in renal cell carcinoma; also, its low expression correlates with poor clinical outcomes in renal cell carcinoma. Together, our study not only identifies FILNC1 as a negative regulator of renal cancer with potential clinical value, but also reveals a regulatory mechanism by long non-coding RNAs to control energy metabolism and tumor development.FoxO are commonly down-regulated transcription factors and tumor suppressors in renal cell cancer (RCC). Here, the authors show that upon energy stress FoxOs induce the expression of the long non-coding RNA FILNC1, which inhibits survival of RCC by downregulating c-Myc and c-Myc-dependent metabolic rewiring.

BAP1 inhibits the ER stress gene regulatory network and modulates metabolic stress response.

The endoplasmic reticulum (ER) is classically linked to metabolic homeostasis via the activation of unfolded protein response (UPR), which is instructed by multiple transcriptional regulatory cascades. BRCA1 associated protein 1 (BAP1) is a tumor suppressor with de-ubiquitinating enzyme activity and has been implicated in chromatin regulation of gene expression. Here we show that BAP1 inhibits cell death induced by unresolved metabolic stress. This prosurvival role of BAP1 depends on its de-ubiquitinating activity and correlates with its ability to dampen the metabolic stress-induced UPR transcriptional network. BAP1 inhibits glucose deprivation-induced reactive oxygen species and ATP depletion, two cellular events contributing to the ER stress-induced cell death. In line with this, Bap1 KO mice are more sensitive to tunicamycin-induced renal damage. Mechanically, we show that BAP1 represses metabolic stress-induced UPR and cell death through activating transcription factor 3 (ATF3) and C/EBP homologous protein (CHOP), and reveal that BAP1 binds to ATF3 and CHOP promoters and inhibits their transcription. Taken together, our results establish a previously unappreciated role of BAP1 in modulating the cellular adaptability to metabolic stress and uncover a pivotal function of BAP1 in the regulation of the ER stress gene-regulatory network. Our study may also provide new conceptual framework for further understanding BAP1 function in cancer.

NBR2: A former junk gene emerges as a key player in tumor suppression.

NBR2 (neighbor of BRCA1 gene 2) is a non-protein coding gene that resides adjacent to tumor suppressor gene BRCA1, but its role in cancer biology has remained unknown. Our recent study showed that NBR2 encodes a long non-coding RNA and suppresses tumor development through regulation of adenosine monophosphate-activated protein kinase (AMPK) activation.

Long non-coding RNAs in cancer metabolism.

Altered cellular metabolism is an emerging hallmark of cancer. Accumulating recent evidence links long non-coding RNAs (lncRNAs), a still poorly understood class of non-coding RNAs, to cancer metabolism. Here we review the emerging findings on the functions of lncRNAs in cancer metabolism, with particular emphasis on how lncRNAs regulate glucose and glutamine metabolism in cancer cells, discuss how lncRNAs regulate various aspects of cancer metabolism through their cross-talk with other macromolecules, explore the mechanistic conceptual framework of lncRNAs in reprogramming metabolism in cancers, and highlight the challenges in this field. A more in-depth understanding of lncRNAs in cancer metabolism may enable the development of novel and effective therapeutic strategies targeting cancer metabolism.

BAF180 regulates cellular senescence and hematopoietic stem cell homeostasis through p21.

BAF180 (also called PBRM1), a subunit of the SWI/SNF complex, plays critical roles in the regulation of chromatin remodeling and gene transcription, and is frequently mutated in several human cancers. However, the role of mammalian BAF180 in tumor suppression and tissue maintenance in vivo remains largely unknown. Here, using a conditional somatic knockout approach, we explored the cellular and organismal functions of BAF180 in mouse. BAF180 deletion in primary mouse embryonic fibroblasts (MEFs) triggers profound cell cycle arrest, premature cellular senescence, without affecting DNA damage response or chromosomal integrity. While somatic deletion of BAF180 in adult mice does not provoke tumor development, BAF180 deficient mice exhibit defects in hematopoietic system characterized by progressive reduction of hematopoietic stem cells (HSCs), defective long-term repopulating potential, and hematopoietic lineage developmental aberrations. BAF180 deletion results in elevated p21 expression in both MEFs and HSCs. Mechanistically, we showed that BAF180 binds to p21 promoter, and BAF180 deletion enhances the binding of modified histones associated with transcriptional activation on p21 promoter. Deletion of p21 rescues cell cycle arrest and premature senescence in BAF180 deficient MEFs, and partially rescues hematopoietic defects in BAF180 deficient mice. Together, our study identifies BAF180 as a critical regulator of cellular senescence and HSC homeostasis, which is at least partially regulated through BAF180-mediated suppression of p21 expression. Our results also suggest that senescence triggered by BAF180 inactivation may serve as a failsafe mechanism to restrain BAF180 deficiency-associated tumor development, providing a conceptual framework to further understand BAF180 function in tumor biology.

LncRNA NBR2 engages a metabolic checkpoint by regulating AMPK under energy stress.

Long non-coding RNAs (lncRNAs) have emerged as critical regulators in various cellular processes. However, the potential involvement of lncRNAs in kinase signalling remains largely unknown. AMP-activated protein kinase (AMPK) acts as a critical sensor of cellular energy status. Here we show that the lncRNA NBR2 (neighbour of BRCA1 gene 2) is induced by the LKB1-AMPK pathway under energy stress. On energy stress, NBR2 in turn interacts with AMPK and promotes AMPK kinase activity, thus forming a feed-forward loop to potentiate AMPK activation during energy stress. Depletion of NBR2 attenuates energy-stress-induced AMPK activation, resulting in unchecked cell cycling, altered apoptosis/autophagy response, and increased tumour development in vivo. NBR2 is downregulated and its low expression correlates with poor clinical outcomes in some human cancers. Together, the results of our study uncover a mechanism coupling lncRNAs with metabolic stress response, and provides a broad framework to understand further the regulation of kinase signalling by lncRNAs.

AMPK modulates Hippo pathway activity to regulate energy homeostasis.

The Hippo pathway was discovered as a conserved tumour suppressor pathway restricting cell proliferation and apoptosis. However, the upstream signals that regulate the Hippo pathway in the context of organ size control and cancer prevention are largely unknown. Here, we report that glucose, the ubiquitous energy source used for ATP generation, regulates the Hippo pathway downstream effector YAP. We show that both the Hippo pathway and AMP-activated protein kinase (AMPK) were activated during glucose starvation, resulting in phosphorylation of YAP and contributing to its inactivation. We also identified glucose-transporter 3 (GLUT3) as a YAP-regulated gene involved in glucose metabolism. Together, these results demonstrate that glucose-mediated energy homeostasis is an upstream event involved in regulation of the Hippo pathway and, potentially, an oncogenic function of YAP in promoting glycolysis, thereby providing an exciting link between glucose metabolism and the Hippo pathway in tissue maintenance and cancer prevention.

Liver-enriched transcription factors regulate microRNA-122 that targets CUTL1 during liver development.

UNLABELLED: MicroRNA-122 (miR-122) is a liver-specific microRNA whose expression is specifically turned on in the mouse liver during embryogenesis, thus it is expected to be involved in liver development. However, the role of miR-122 in liver development and its potential underlying mechanism remain unclear. Here, we show that the expression of miR-122 is closely correlated with four liver-enriched transcription factors (LETFs)-hepatocyte nuclear factor (HNF) 1α, HNF3ß, HNF4α, and CCAAT/enhancer-binding protein (C/EBP) α-in the livers of developing mouse embryos and in human hepatocellular carcinoma (HCC) cell lines. Correspondingly, promoter analysis revealed that these LETFs are coordinately involved in the transcriptional regulation of miR-122, and three HNFs directly bind to the miR-122 promoter as transcriptional activators. Using a luciferase reporter system, we identified a group of miR-122 targets involved in proliferation and differentiation regulation. Among these targets, the most prominently repressed target was CUTL1, a transcriptional repressor of genes specifying terminal differentiation in multiple cell lineages, including hepatocytes. We show that CUTL1 expression is gradually silenced at the posttranscriptional level during mouse liver development. Overexpression and knockdown studies both showed that miR-122 repressed CUTL1 protein expression in HCC cell lines. Finally, we show that the stable restoration of miR-122 in HepG2 cells suppresses cellular proliferation and activates the expression of three hepatocyte functional genes, including the cholesterol-7α hydroxylase gene (CYP7A1), a known target of CUTL1 in hepatocytes. CONCLUSION: Our study provides a model in which miR-122 functions as an effector of LETFs and contributes to liver development by regulating the balance between proliferation and differentiation of hepatocytes, at least by targeting CUTL1.

[Clinical significance of serum levels of endostatin in patients with clear cell renal cell carcinoma].

OBJECTIVE: To assess serum levels of endogenous endostatin in patients with clear cell renal cell carcinoma (CCRCC) and to determine the relationship of these levels to tumor stage, grade. METHODS: From March 2004 to October 2008, preoperative serum were obtained from 138 consecutive patients with CCRCC (73 patients in T1, 39 patients in T2, 20 patients in T3, and 6 patients in T4) and 40 healthy controls. Serum levels of endostatin were measured by sandwich-ELISA. Associations between circulating endostatin levels and clinicopathologic factors and clinical outcome were determined. RESULTS: Endostatin levels did not differ significantly between the patients with CCRCC (93.1 microg/L) and healthy controls (78.9 microg/L, P > 0.05). Serum levels of endostatin were significantly higher in the T2-4 CCRCC patients (107.2 microg/L) than those of the T1 patients (80.4 microg/L, P < 0.01). No significant difference was found in the endostatin levels among the T2-4 patients, or between healthy controls and the T1 patients. The serum endostatin concentration was significantly higher in the metastasis group (118.4 microg/L) than in the no metastasis group (89.5 microg/L, P < 0.05), but there was no significant difference between patients with distant metastasis group (122.0 microg/L) and lymph nodes metastasis (110.0 microg/L, P > 0.05). Patients with G3-4 tumors had significantly higher endostatin levels (111.8 microg/L) than those of patients with G1 (80.4 microg/L) and G2 tumors (86.2 microg/L, P < 0.01), but endostatin levels did not differ significantly between the two groups (P > 0.05). CONCLUSION: Preoperative serum levels of endostatin elevated in patients with CCRCC and associated with higher stage and grade.

[Urachal mass in adults: clinical analysis of 33 cases].

OBJECTIVE: To summarize the experience in diagnosis and treatment of urachal mass in adults. METHODS: The clinical data of 33 patients with urachal mass, 21 male and 12 females, aged 49 (30-75), were analyzed retrospectively. RESULTS: The urachal masses of 11 patients (33%, 11/33) were benign, including 5 cases of abscess, 3 of cyst, 2 of malacoplakia, and 1 of xanthogranuloma. The most common symptom of the benign urachal mass was abdominal mass (36%). Seven patients underwent computed tomography (CT) and no calcification was found. All 11 patients were treated successfully by mass excision. Twenty-two cases (67%) had urachal carcinomas, including 17 cases of adenocarcinoma, 4 of squamous carcinoma, and 1 of transitional cell carcinoma. The most common symptom was gross hematuria (68%) in the malignant urachal mass patients and CT showed calcification in 38% of them (5/13). The overall 5-year cancer-specific survival rate was 45.2%. Nine patients with localized cancer underwent extended partial cystectomy with a 5-year cancer-specific survival rate of 78.3%. The patients with metastatic urachal cancer underwent multimodal treatment with an objective response rate of 50% for chemotherapy and 25% for radiation therapy. The median survival time after metastasis of the 8 cases given multimodal treatment was 14.0 months, 7.5 months longer than that of the 4 cases untreated (6.5 months, P = 0.09). CONCLUSION: The majority of urachal mass in adults is malignant. CT helps in distinguishing the benignancy from malignancy of urachal mass. Surgical excision affords good course for benign urachal mass. Extended partial cystectomy provides a curative surgical treatment for localized urachal cancer. The prognosis of metastatic urachal cancer is poor. Active multimodal treatment may improve the survival of patients with metastatic disease.