HDAC6 Suppresses Let-7i-5p to Elicit TSP1/CD47-Mediated Anti-Tumorigenesis and Phagocytosis of Hepatocellular Carcinoma.

Histone deacetylase 6 (HDAC6) uniquely serves as a tumor suppressor in hepatocellular carcinogenesis, but the underlying mechanisms leading to tumor suppression are not fully understood. To identify comprehensive microRNAs (miRNAs) regulated by HDAC6 in hepatocellular carcinogenesis, differential miRNA expression analysis of HDAC6-transfected Hep3B cells was performed. Using integrative analyses of publicly available transcriptome data and miRNA target prediction, we selected five candidate miRNAs and, through in vitro functional validation, showed that let-7i-5p specifically suppressed thrombospondin-1 (TSP1) in hepatocellular carcinoma (HCC). Ectopic expression of antisense let-7i-5p (AS-let-7i-5p) inhibited in vitro tumorigenesis of HCC cells. In addition, treatments of partially purified TSP1 from culture cell media (ppTSP1) and recombinant TSP1 (rTSP1) exhibited similar effects with AS-let-7i-5p treatment on the same HCC cells, whereas TSP1 neutralizing antibody treatment significantly attenuated these effects. Notably, treatments of HDAC6 plasmid, AS-let-7i-5p, ppTSP1, and rTSP1 significantly suppressed in vitro angiogenesis and metastatic potential of HCC cells, but the co-treatment of TSP1 antibody specific to cluster of differentiation 47 (CD47) binding domain successfully blocked these effects in the same cells. Furthermore, we demonstrated that recovery of HDAC6 elicited let-7i-5p suppression to de-repress TSP1 expression; therefore, it occupied the CD47 receptor to block CD47-SIRPα-mediated anti-phagocytosis of macrophage in HCC. We also observed that HCC-derived exosomal let-7i-5p suppressed TSP1 of recipient hepatocyte cells. Treatments of HDAC6 plasmid, AS-let-7i-5p, and rTSP1 suppressed tumor incidence as well as tumor growth rates in a spontaneous mouse HCC model. Conclusion: Our findings suggest that the HDAC6-let-7i-5p-TSP1 regulatory pathway suppresses neoplastic and antiphagocytic behaviors of HCC by interacting with cell surface receptor CD47 in HCC and neighboring cells of tumor microenvironment, providing a therapeutic target for the treatment of liver malignancy and metastasis.

Barrier to autointegration factor 1, procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3, and splicing factor 3b subunit 4 as early-stage cancer decision markers and drivers of hepatocellular carcinoma.

An accurate tool enabling early diagnosis of hepatocellular carcinoma (HCC) is clinically important, given that early detection of HCC markedly improves survival. We aimed to investigate the molecular markers underlying early progression of HCC that can be detected in precancerous lesions. We designed a gene selection strategy to identify potential driver genes by integrative analysis of transcriptome and clinicopathological data of human multistage HCC tissues, including precancerous lesions, low- and high-grade dysplastic nodules. The gene selection process was guided by detecting the selected molecules in both HCC and precancerous lesion. Using various computational approaches, we selected 10 gene elements as a candidate and, through immunohistochemical staining, showed that barrier to autointegration factor 1 (BANF1), procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (PLOD3), and splicing factor 3b subunit 4 (SF3B4) are HCC decision markers with superior capability to diagnose early-stage HCC in a large cohort of HCC patients, as compared to the currently popular trio of HCC diagnostic markers: glypican 3, glutamine synthetase, and heat-shock protein 70. Targeted inactivation of BANF1, PLOD3, and SF3B4 inhibits in vitro and in vivo liver tumorigenesis by selectively modulating epithelial-mesenchymal transition and cell-cycle proteins. Treatment of nanoparticles containing small-interfering RNAs of the three genes suppressed liver tumor incidence as well as tumor growth rates in a spontaneous mouse HCC model. We also demonstrated that SF3B4 overexpression triggers SF3b complex to splice tumor suppressor KLF4 transcript to nonfunctional skipped exon transcripts. This contributes to malignant transformation and growth of hepatocyte through transcriptional inactivation of p27Kip1 and simultaneously activation of Slug genes. CONCLUSION: The findings suggest molecular markers of BANF1, PLOD3, and SF3B4 indicating early-stage HCC in precancerous lesion, and also suggest drivers for understanding the development of hepatocarcinogenesis. (Hepatology 2018;67:1360-1377).

MicroRNA-495-3p functions as a tumor suppressor by regulating multiple epigenetic modifiers in gastric carcinogenesis.

MicroRNAs (miRNAs) engage in complex interactions with the machinery that controls the transcriptome and concurrently target multiple mRNAs. Here, we demonstrate that microRNA-495-3p (miR-495-3p) functions as a potent tumor suppressor by governing ten oncogenic epigenetic modifiers (EMs) in gastric carcinogenesis. From the large cohort transcriptome datasets of gastric cancer (GC) patients available from The Cancer Genome Atlas (TCGA) and the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO), we were able to recapitulate 15 EMs as significantly overexpressed in GC among the 51 EMs that were previously reported to be involved in cancer progression. Computational target prediction yielded miR-495-3p, which targets as many as ten of the 15 candidate oncogenic EMs. Ectopic expression of miRNA mimics in GC cells caused miR-495-3p to suppress ten EMs, and inhibited tumor cell growth and proliferation via caspase-dependent and caspase-independent cell death processing. In addition, in vitro metastasis assays showed that miR-495-3p plays a role in the metastatic behavior of GC cells by regulating SLUG, vimentin, and N-cadherin. Furthermore, treatment of GC cells with 5-aza-2'-deoxcytidine restored miR-495-3p expression; sequence analysis revealed hypermethylation of the miR-495-3p promoter region in GC cells. A negative regulatory loop is proposed, whereby DNMT1, among ten oncogenic EMs, regulates miR-495-3p expression via hypermethylation of the miR-495-3p promoter. Our findings suggest that the functional loss or suppression of miR-495-3p triggers overexpression of multiple oncogenic EMs, and thereby contributes to malignant transformation and growth of gastric epithelial cells. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The roles and mechanisms of coding and noncoding RNA variations in cancer.

Functional variations in coding and noncoding RNAs are crucial in tumorigenesis, with cancer-specific alterations often resulting from chemical modifications and posttranscriptional processes mediated by enzymes. These RNA variations have been linked to tumor cell proliferation, growth, metastasis, and drug resistance and are valuable for identifying diagnostic or prognostic cancer biomarkers. The diversity of posttranscriptional RNA modifications, such as splicing, polyadenylation, methylation, and editing, is particularly significant due to their prevalence and impact on cancer progression. Additionally, other modifications, including RNA acetylation, circularization, miRNA isomerization, and pseudouridination, are recognized as key contributors to cancer development. Understanding the mechanisms underlying these RNA modifications in cancer can enhance our knowledge of cancer biology and facilitate the development of innovative therapeutic strategies. Targeting these RNA modifications and their regulatory enzymes may pave the way for novel RNA-based therapies, enabling tailored interventions for specific cancer subtypes. This review provides a comprehensive overview of the roles and mechanisms of various coding and noncoding RNA modifications in cancer progression and highlights recent advancements in RNA-based therapeutic applications.

ADAR1-dependent miR-3144-3p editing simultaneously induces MSI2 expression and suppresses SLC38A4 expression in liver cancer.

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 < G)) in liver cancer cells. We then demonstrated that 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 < G). The ectopic expression of both ADAR1 and the ED_miR-3144-3p(3_A < G) mimic enhanced mitotic activities, and ADAR1 suppressed SLC38A4 expression in liver cancer cells. Treatments with mouse-specific ADAR1-, MSI2-siRNA-, or SLC38A4-expressing plasmids suppressed tumorigenesis and tumor growth in a mouse model of spontaneous liver cancer. Our findings suggest that the aberrant regulation of ADAR1 augments oncogenic MSI2 effects by excessively editing canonical miR-3144-3p and that the resultant ED_miR-3144-3p(3_A < G) simultaneously suppresses tumor suppressor SLC38A4 expression, contributing to hepatocellular carcinogenesis.

Serum Proteins, HMMR, NXPH4, PITX1 and THBS4; A Panel of Biomarkers for Early Diagnosis of Hepatocellular Carcinoma.

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.

ANKRD13a controls early cell-death checkpoint by interacting with RIP1 independent of NF-κB.

In TNF signaling, ubiquitination of RIP1 functions as an early cell-death checkpoint, which prevents the spatial transition of the signaling complex from complex-I to death-inducing complex-II. Here, we report that ankyrin repeat domain 13a (ANKRD13a) acts as a novel component of complex-II to set a higher signal threshold for the cytotoxic potential of TNF. ANKRD13a deficiency is sufficient to turn the response to TNF from survival to death by promoting the formation of complex-II without affecting NF-κB activation. ANKRD13a binds to ubiquitinated-RIP1 via its UIM, and subsequently limits the association of FADD and caspase-8 with RIP1. Moreover, high ANKRD13a expression is inversely correlated with apoptotic phenotypes in ovarian cancer tissues and is associated with poor prognosis. Our work identifies ANKRD13a as a novel gatekeeper of the early cell-death checkpoint, which may function as part of an escape mechanism from cell death in some cancers.

SMARCA4 oncogenic potential via IRAK1 enhancer to activate Gankyrin and AKR1B10 in liver cancer.

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.

T-cell immune regulator 1 enhances metastasis in hepatocellular carcinoma.

Recurrence and metastasis are major challenges in the management of hepatocellular carcinoma (HCC) patients after resection. To identify a metastasis-associated gene signature, we performed comparative gene expression analysis with recurrent HCC tissues from HCC patients who underwent partial or total hepatectomy and from non-metastatic primary HCC tissues. From this, we were able to identify genes associated with HCC recurrence. TCIRG1 (T-Cell Immune Regulator 1) was one of the aberrantly overexpressed genes in patients with recurrent HCC who had undergone total hepatectomy. The significant overexpression of TCIRG1 was confirmed using the Liver Hepatocellular Carcinoma dataset from The Cancer Genome Atlas. High expression of TCIRG1 was significantly associated with poor 5-year disease-free and recurrence-free survival of HCC patients. TCIRG1 knockdown suppressed tumor cell growth and proliferation in HCC cell lines; caused a significant increase in the proportion of cells in the G1/S phase of cell cycle; induced cell death; suppressed the metastatic potential of HCC cells by selectively regulating the epithelial-mesenchymal transition (EMT) regulatory proteins E-cadherin, N-cadherin, Fibronectin, Snail and Slug; and significantly attenuated the metastatic potential of ras-transformed NIH-3T3 cells in vitro and in vivo. These findings suggest that TCIRG1 functions as a metastatic enhancer by modulating growth, death and EMT in HCC cells. TCIRG1 could be a therapeutic target for the treatment of liver malignancy and metastasis.