Search

Aberrant adenosine-to-inosine (A-to-I) RNA editing, catalyzed by adenosine deaminase acting on double-stranded RNA (ADAR), has been implicated in various cancers, but the mechanisms by which microRNA (miRNA) editing contributes to cancer development are largely unknown. Our multistage hepatocellular carcinogenesis transcriptome data analyses, together with publicly available data, indicated that ADAR1 was the most profoundly dysregulated gene among RNA-editing enzyme family members in liver cancer. Targeted inactivation of ADAR1 inhibited the in vitro tumorigenesis of liver cancer cells. An integrative computational analyses of RNA-edited hotspots and the known editing frequency of miRNAs suggested that the miRNA miR-3144-3p was edited by ADAR1 during liver cancer progression. Specifically, ADAR1 promoted A-to-I editing of canonical miR-3144-3p to replace the adenosine at Position 3 in the seed region with a guanine (ED_miR-3144-3p(3_A Musashi RNA-binding protein 2 (MSI2) was a specific target of miR-3144-3p and that MSI2 overexpression was due to excessive ADAR1-dependent over-editing of canonical miR-3144-3p in liver cancer. In addition, target prediction analyses and validation experiments identified solute carrier family 38 member 4 (SLC38A4) as a specific gene target of ED_miR-3144-3p(3_A

Aberrant adenosine-to-inosine (A-to-I) RNA editing, catalyzed by adenosine deaminase acting on double-stranded RNA (ADAR), is implicated in various cancers, but the mechanisms by which microRNA (miRNA) editing contributes to cancer development are currently largely unknown. Our multi-step hepatocellular carcinogenesis transcriptome data analyses, together with publicly available data, indicated that ADAR1 is the most dysregulated gene among the RNA editing enzyme families in liver cancer. Targeted inactivation of ADAR1 inhibits in vitro tumorigenesis of liver cancer cells. Integrative computational analyses of RNA editing hotspots and the editing frequency of miRNAs suggested miR-3144-3p a potential mRNA edited by ADAR1 in liver cancer progression. ADAR1 promoted A-to-I editing of the canonical miR-3144-3p to change position 3 adenosine in the seed region to guanine (ED_miR-3144-3p(3_A Musashi RNA-binding protein 2 (MSI2) is a specific target of miR-3144-3p, and that MSI2 overexpression is due to ADAR1-dependent over-editing of the canonical miR-3144-3p in liver cancer. In addition, target prediction analyses and validation experiments identified solute carrier family 38 member 4 (SLC38A4) as specific target gene for ED_miR-3144-3p(3_A

Background: Aberrant adenosine-to-inosine (A-to-I) RNA editing, catalyzed by adenosine deaminase acting on double-stranded RNA (ADAR), is implicated in various cancers, but the mechanisms by which microRNA (miRNA) editing contributes to cancer development are currently largely unknown.Method: Our multi-step hepatocellular carcinogenesis transcriptome data analyses, together with publicly available data, indicated that ADAR1 is the most dysregulated gene among the RNA editing enzyme families in hepatocellular carcinoma (HCC). Targeted inactivation of ADAR1 inhibits in vitro tumorigenesis of HCC cells. Integrative computational analyses of RNA editing hotspots and the editing frequency of miRNAs suggested miR-3144-3p a potential mRNA edited by ADAR1 in hepatocellular carcinogenesis.Result: Through direct sequencing of ADAR1-knocked-down or -overexpressing cells, we demonstrated that ADAR1 promoted A-to-I editing of the canonical miR-3144-3p to change position 3 adenosine in the seed region to guanine (ED_miR-3144-3p(3_AMusashi RNA-binding protein 2 (MSI2) is a specific target of miR-3144-3p, and that MSI2 overexpression is due to ADAR1-dependent over-editing of the canonical miR-3144-3p in HCC cells. Notably miR-3144-3p treatments showed similar to in vitro anti-tumor activities of ADAR1-specifc siRNA in HCC. In addition, target prediction analyses and validation experiments identified solute carrier family 38 member 4 (SLC38A4) as specific target gene for ED_miR-3144-3p(3_AConclusions: Our findings suggest that aberrant regulation of ADAR1 augments oncogenic MSI2 via overediting the canonical miR-3144-3p, and the resultant ED_miR-3144-3p(3_A

Previously, we demonstrated that recovery of Histone deacetylase 6 (HDAC6) elicited let-7i-5p suppression to de-repress the thrombospondin-1 (TSP1) expression, and thereby it occupied CD47 receptor to block CD47-SIRPα mediated anti-phagocytosis of macrophage in liver cancer. NRT-YHD_001 is a modified antisense miRNA of let-7i-5p for liver cancer treatment. NRT-YHD_001 was substituted with O-methoxyethyl and phosphothioate for stability, and N-acetylgalactosamine was attached for specific delivery to the liver. In in vitro phagocytosis assay, NRT-YHD_001 showed macrophage phagocytosis activity with similar or better results compared to non-modified antisense let-7i-5p (AS_let-7i-5p). In addition, it was confirmed that NRT-YHD_001 inhibited endogenous let-7i-5p of liver cancer cells and activated macrophage phagocytosis at 4°C or 25°C for up to 2 weeks. When NRT-YHD_001 activity was investigated in vivo after 1 week, 2 weeks, and 4 weeks after administration of single dose NRT-YHD_001 to the tail vein of a mouse, NRT-YHD_001 was not degraded, and let-7i-5p was inhibited while maintaining activity in liver tissue for 2 weeks after injection. To investigate the in vivo therapeutic effect, a mouse spontaneous (Ras-transgenic) liver cancer model was used and compared with the sorafenib-administered group. After administration of NRT-YHD_001 and sorafenib each week at the time when liver mass was detected, the tumor size and growth rate were compared. As a result, the NRT-YHD_001 administration group showed significant therapeutic effect compared to sorafenib. These results showed that NRT-YHD_001 had a strong anticancer effect by converting the macrophage of don't eat me into eat me signal in the tumor microenvironment of liver cancer, thereby removing or inhibiting liver cancer cells. Keywords: let-7i-5p, macrophage, phagocytosis, liver cancer, NRT-YHD_001 Citation Format: Suk Woo Nam, Sangyean Kim, Min Jeong Na, Eunbi Shin, Jin Woong Ha, Soyoung Jeon, Jeong Hwan Yoon. In vivo therapeutic validation of NRT-YHD_001, a novel macrophage checkpoint inhibitor, in liver cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5165.

The high morbidity rate of hepatocellular carcinoma (HCC) is mainly linked to late diagnosis. Early diagnosis of this leading cause of mortality is therefore extremely important. We designed a gene selection strategy to identify potential secretory proteins by predicting signal peptide cleavage sites in amino acid sequences derived from transcriptome data of human multistage HCC comprising chronic hepatitis, liver cirrhosis and early and overt HCCs. The gene selection process was validated by the detection of molecules in the serum of HCC patients. From the computational approaches, 10 gene elements were suggested as potent candidate secretory markers for detecting HCC patients. ELISA testing of serum showed that hyaluronan mediated motility receptor (HMMR), neurexophilin 4 (NXPH4), paired like homeodomain 1 (PITX1) and thrombospondin 4 (THBS4) are early-stage HCC diagnostic markers with superior predictive capability in a large cohort of HCC patients. In the assessment of differential diagnostic accuracy, receiver operating characteristic curve analyses showed that HMMR and THBS4 were superior to α-fetoprotein (AFP) in diagnosing HCC, as evidenced by the high area under the curve, sensitivity, specificity, accuracy and other values. In addition, comparative analysis of all four markers and AFP combinations demonstrated that HMMR-PITX1-AFP and HMMR-NXPH4-PITX1 trios were the optimal combinations for reaching 100% accuracy in HCC diagnosis. Serum proteins HMMR, NXPH4, PITX1 and THBS4 can complement measurement of AFP in diagnosing HCC and improve identification of patients with AFP-negative HCC as well as discriminate HCC from non-malignant chronic liver disease.

SWItch/Sucrose Non-Fermentable (SWI/SNF) is a multiprotein complex essential for the regulation of eukaryotic gene expression. SWI/SNF complex genes are genetically altered in over 20% of human malignancies, but the aberrant regulation of the SWI/SNF subunit genes and subsequent dysfunction caused by abnormal expression of subunit gene in cancer, remain poorly understood. Among the SWI/SNF subunit genes, SMARCA4, SMARCC1, and SMARCA2 were identified to be overexpressed in human hepatocellular carcinoma (HCC). Modulation of SMARCA4, SMARCC1, and SMARCA2 inhibited in vitro tumorigenesis of HCC cells. However, SMARCA4-targeting elicited remarkable inhibition in an in vivo Ras-transgenic mouse HCC model (Ras-Tg), and high expression levels of SMARCA4 significantly associated with poor prognosis in HCC patients. Furthermore, most HCC patients (72-86%) showed SMARCA4 overexpression compared to healthy controls. To identify SMARCA4-specific active enhancers, mapping, and analysis of chromatin state in liver cancer cells were performed. Integrative analysis of SMARCA4-regulated genes and active chromatin enhancers suggested 37 genes that are strongly activated by SMARCA4 in HCC. Through chromatin immunoprecipitation-qPCR and luciferase assays, we demonstrated that SMARCA4 activates Interleukin-1 receptor-associated kinase 1 (IRAK1) expression through IRAK1 active enhancer in HCC. We then showed that transcriptional activation of IRAK1 induces oncoprotein Gankyrin and aldo-keto reductase family 1 member B10 (AKR1B10) in HCC. The regulatory mechanism of the SMARCA4-IRAK1-Gankyrin, AKR1B10 axis was further demonstrated in HCC cells and in vivo Ras-Tg mice. Our results suggest that aberrant overexpression of SMARCA4 causes SWI/SNF to promote IRAK1 enhancer to activate oncoprotein Gankyrin and AKR1B10, thereby contributing to hepatocarcinogenesis.

Arginine residues in a protein can be enzymatically deiminated to citrulline residues, and this post-translational modification of proteins, called citrullination, has been known to be associated with autoimmune diseases such as rheumatoid arthritis. Since citrullination of an arginine residue causes a mass shift of only 1 Da, isotopic distribution patterns of citrullinated and its control (non-citrullinated) peptides overlap each other, and the resulting isotopic pattern is skewed with respect to that of the control peptide. Recently, Deforce and coworkers utilized the citrullination-induced skewed isotopic patterns for estimating the extent of citrullination (J. Proteome Res. 2012, 11, 5245). However, electrospray ionization (ESI), which Deforce and coworkers employed, is not fully suitable for this type of quantification because the number of potential charge sites on a citrullinated peptide is less than that on its control peptide and this leads to different ion abundances for a given charge state. In this study, we employed matrix-assisted laser desorption/ionization (MALDI) instead of ESI for quantification of citrullination because the preference for singly charged ion formation by MALDI may overcome the aforementioned issue. Our results show that the isotopic patterns generated by MALDI time-of-flight (TOF) mass spectrometry (MS) reflect the citrullination contents better than those generated by ESI linear ion trap (LIT) MS.