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Table S1-Summary of HCC clinical cohorts and their omics datasets. Table S2-Primers and oligoes used in this study

The lower level of miR-192-5p was associated with poor prognosis of HCC patients.

Various cancer stem cell (CSC) biomarkers have been identified for hepatocellular carcinoma (HCC), but little is known about the implications of heterogeneity and shared molecular networks within the CSC population. Through miRNA profile analysis in an HCC cohort (n = 241) for five groups of CSC+ HCC tissues, i.e., EpCAM+, CD90+, CD133+, CD44+, and CD24+ HCC, we identified a 14-miRNA signature commonly altered among these five groups of CSC+ HCC. miR-192-5p, the top-ranked CSC miRNA, was liver-abundant and -specific and markedly downregulated in all five groups of CSC+ HCC from two independent cohorts (n = 613). Suppressing miR-192-5p in HCC cells significantly increased multiple CSC populations and CSC-related features through targeting PABPC4. Both TP53 mutation and hypermethylation of the mir-192 promoter impeded transcriptional activation of miR-192-5p in HCC cell lines and primary CSC+ HCC. This study reveals the circuit from hypermethylation of the mir-192 promoter through the increase in PABPC4 as a shared genetic regulatory pathway in various groups of primary CSC+ HCC. This circuit may be the driver that steers liver cells toward hepatic CSC cells, leading to hepatic carcinogenesis.Significance:miR-192-5p and its regulatory pathway is significantly abolished in multiple groups of HCC expressing high levels of CSC markers, which may represent a key event for hepatic carcinogenesis.

It includes additional materials and methods, i.e., Plasmids, siRNAs, and LY294002 treatment; assays to examine cell malignancy features; qPCR and Western blot.

Mechanistic study and precision treatment of primary liver cancer (PLC) are hindered by marked heterogeneity, which is challenging to recapitulate in any given liver cancer mouse model. Here, we report the generation of 25 mouse models of PLC by in situ genome editing of hepatocytes recapitulating 25 single or combinations of human cancer driver genes. These mouse tumors represent major histopathological types of human PLCs and could be divided into three human-matched molecular subtypes based on transcriptomic and proteomic profiles. Phenotypical characterization identified subtype- or genotype-specific alterations in immune microenvironment, metabolic reprogramming, cell proliferation, and expression of drug targets. Furthermore, single-cell analysis and expression tracing revealed spatial and temporal dynamics in expression of pyruvate kinase M2 ( Pkm2 ). Tumor-specific knockdown of Pkm2 by multiplexed genome editing reversed the Warburg effect and suppressed tumorigenesis in a genotype-specific manner. Our study provides mouse PLC models with defined genetic drivers and characterized phenotypical heterogeneity suitable for mechanistic investigation and preclinical testing.

Background and aims Transarterial chemoembolization (TACE) is a standard locoregional therapy for patients with hepatocellular carcinoma (HCC) patients with a variable overall response in efficacy. We aimed to identify key molecular signatures and related pathways leading to HCC resistance to TACE, with the hope of developing effective approaches in preselecting patients with survival benefit from TACE. Approach and results Four independent HCC cohorts with 680 patients were used. MicroRNA (miRNA) transcriptome analysis in patients with HCC revealed a 41-miRNA signature related to HCC recurrence after adjuvant TACE, and miR-125b was the top reduced miRNA in patients with HCC recurrence. Consistently, patients with HCC with low miR-125b expression in tumor had significantly shorter time to recurrence following adjuvant TACE in two independent cohorts. Loss of miR-125b in HCC noticeably activated the hypoxia inducible factor 1 alpha subunit (HIF1α)/pAKT loop in vitro and in vivo. miR-125b directly attenuated HIF1α translation through binding to HIF1A internal ribosome entry site region and targeting YB-1, and blocked an autocrine HIF1α/platelet-derived growth factor β (PDGFβ)/pAKT/HIF1α loop of HIF1α translation by targeting the PDGFβ receptor. The miR-125b-loss/HIF1α axis induced the expression of CD24 and erythropoietin (EPO) and enriched a TACE-resistant CD24-positive cancer stem cell population. Consistently, patients with high CD24 or EPO in HCC had poor prognosis following adjuvant TACE therapy. Additionally, in patients with HCC having TACE as their first-line therapy, high EPO in blood before TACE was also noticeably related to poor response to TACE. Conclusions MiR-125b loss activated the HIF1α/pAKT loop, contributing to HCC resistance to TACE and the key nodes in this axis hold the potential in assisting patients with HCC to choose TACE therapy.

The test for the cancer stem cell (CSC) hypothesis is to find a target expressed on all, and only CSCs in a patient tumor, then eliminate all cells with that target that eliminates the cancer. That test has not yet been achieved, but CSC diagnostics and targets found on CSCs and some other cells have resulted in a few clinically relevant therapies. However, it has become apparent that eliminating the subset of tumor cells characterized by self-renewal properties is essential for long-term tumor control. CSCs are able to regenerate and initiate tumor growth, recapitulating the heterogeneity present in the tumor before treatment. As great progress has been made in identifying and elucidating the biology of CSCs as well as their interactions with the tumor microenvironment, the time seems ripe for novel therapeutic strategies that target CSCs to find clinical applicability. On May 19-21, 2021, researchers in cancer stem cells met virtually for the Keystone eSymposium "Cancer Stem Cells: Advances in Biology and Clinical Translation" to discuss recent advances in the understanding of CSCs as well as clinical efforts to target these populations.

Transarterial chemoembolization (TACE) is a commonly used treatment modality in hepatocellular carcinoma (HCC). The ability to identify patients who will respond to TACE represents an important clinical need, and tumor gene expression patterns may be associated with TACE response. We investigated whether tumor transcriptome is associated with TACE response in patients with HCC. We analyzed transcriptome data of treatment-naive tumor tissues from a Chinese cohort of 191 HCC patients, including 105 patients who underwent TACE following resection with curative intent. We then developed a gene signature, TACE Navigator, which was associated with improved survival in patients that received either adjuvant or post-relapse TACE. To validate our findings, we applied our signature in a blinded manner to three independent cohorts comprising an additional 130 patients with diverse ethnic backgrounds enrolled in three different hospitals who received either adjuvant TACE or palliative TACE. TACE Navigator stratified patients into Responders and Non-Responders which was associated with improved survival following TACE in our test cohort (Responders: 67 months vs Non-Responders: 39.5 months, p

Background Emerging studies revealed that cancer stem cells (CSCs) possessed peculiar metabolic properties, which however remained largely unknown in hepatocellular carcinoma (HCC). Genetic silencing of liver-abundant miR-192-5p was a key feature for multiple groups of CSC-positive HCCs. We thus aimed to investigate essential metabolic features of hepatic CSCs via using HCCs with miR-192-5p silencing as a model. Methods Datasets from two independent HCC cohorts were used. Data integration analyses of miR-192-5p with metabolome and mRNA transcriptome data in HCC Cohort 1 were performed to investigate miR-192-5p related metabolic features, which was further validated in Cohort 2. Cellular and molecular assays were performed to examine whether and how miR-192-5p regulated the identified metabolic features. Co-culture systems consisting of HCC cells and LX2 (human hepatic stellate cell line) or THP1 (human monocyte cell line) were established to explore effects of the identified metabolic properties on stemness features of HCC cells via interacting with co-cultured non-tumor cells. Results High levels of glycolysis-related metabolites and genes were present in HCCs with low miR-192-5p and CSC-positive HCCs in two independent HCC cohorts. miR-192-5p knockout cells displayed CSC features and miR-192-5p loss led to an enhanced glycolytic phenotype via upregulating three bona fide targets, GLUT1 and PFKFB3 (two glycolytic enzymes) and c-Myc (regulating glycolytic genes’ expression). Meanwhile, c-Myc suppressed miR-192-5p transcription, ensuring a low-miR-192-5p/high-c-Myc loop to maintain hyperglycolysis. Moreover, over-produced lactic acid from hyperglycolytic HCC cells stimulated the ERK phosphorylation of co-cultured LX2 and THP1 non-tumor cells partially via NDRG3 and MCT1, which in turn promoted cell malignancy and stemness of HCC cells. Consistently, HCC patients with low level of miR-192-5p in their tumor tissues and high level of NDRG3 or MCT1 in their non-tumor tissues had the shortest overall survival. Conclusions In CSC-positive HCCs, miR-192-5p loss enhanced glycolysis and over produced lactate might further increase HCC malignant features via interacting with environmental non-tumor cells.

Background The gut-liver axis plays a pivotal role in the pathogenesis of hepatocellular carcinoma (HCC). However, the correlations between the gut microbiome and the liver tumor transcriptome in patients with HCC and the impact of the gut microbiota on clinical outcome are less well-understood. Methods Fecal samples collected from HBV-related HCC patients (n = 113) and healthy volunteers (n = 100) were subjected to 16S rRNA sequencing of the microbiome. After a rigorous selection process, 32 paired tumor and adjacent non-tumor liver tissues from the HCC group were subjected to next-generation sequencing (NGS) RNA-seq. The datasets were analyzed individually and integrated with clinical characteristics for combined analysis using bioinformatics approaches. We further verified the potential of the gut microbiota to predict clinical outcome by a random forest model and a support vector machine model. Results We found that Bacteroides, Lachnospiracea incertae sedis, and Clostridium XIVa were enriched in HCC patients with a high tumor burden. By integrating the microbiome and transcriptome, we identified 31 robust associations between the above three genera and well-characterized genes, indicating possible mechanistic relationships in tumor immune microenvironment. Clinical characteristics and database analysis suggested that serum bile acids may be important communication mediators between these three genera and the host transcriptome. Finally, among these three genera, six important microbial markers associated with tumor immune microenvironment or bile acid metabolism showed the potential to predict clinical outcome (AUC = 81%). Conclusions This study revealed that changes in tumor immune microenvironment caused by the gut microbiota via serum bile acids may be important factors associated with tumor burden and adverse clinical outcome. Gut microbes can be used as biomarkers of clinical features and outcomes, and the microbe-associated transcripts of host tumors can partly explain how gut microbiota promotes HCC pathogenesis.

Additional file 2: Supplementary Table S1. Clinical phenotype information of individuals with HCC. Supplementary Table S2. Taxon annotation of 1296 OTUs from all samples. Supplementary Table S3. Fecal microbial diversity index in all samples. Supplementary Table S4. three hundred and ten OTUs from all samples. Supplementary Table S5. Clinical phenotype information of healthy participants. Supplementary Table S6. Clinical characteristics summary of all enrolled individuals. Supplementary Table S7. Different degree of phylum level (p value) in Healthy Control and HCC Group. Supplementary Table S8. Different degree of genera level (p value) in Healthy Control and HCC Group. Supplementary Table S9. The interrelationship between 16S OTU clusters of at genus level and taxonomic compositions (NCBI Taxonomy ID). Supplementary Table S10. fifty-six OTU-gene pairs filtered by FDR test of Pearson correlation between OTUs and genes. Supplementary Table S11. Pearson correlation-based analysis of clinical characteristics (all values) and gut microbiota (75 OTUs matched to Bacteroides & Lachnospiracea incertae sedis & Clostridium XIVa). Supplementary Table S12. Pearson correlation-based analysis of clinical characteristics (abnormal values) and gut microbiota (75 OTUs matched to Bacteroides & Lachnospiracea incertae sedis & Clostridium XIVa). Supplementary Table S13. Metabolites of gut microbes according to Virtual Metabolic Human database ( http://www.vmh.life ). Supplementary Table S14. The relative abundance of six OUT-markers in each sample. Supplementary Table S15. The corresponding bacterial genera of six OTU-markers. Supplementary Table S16. Stool form scale and stool moisture in all samples. (PDF 1380 kb)

Additional file 1: Fig. S1. Study design and flow diagram. Fig. S2. RNA-seq and data analysis. (A) QC passed reads sequenced by an Illumina HiSeq 2500 (pair-end 150-nucleotide read length) for tumors and adjacent non-tumor liver tissues (n = 32, respectively, student’s t test). (B) Parameter of edgeR: plotBCV() and biological coefficient of variation (BCV) was 0.6114732. CPM, counts per million. Fig. S3. There are no differences in gut microbial diversity between healthy controls (n = 100) and HCC patients (n = 113). (A) Shannon-Wiener curves for all 213 fecal samples. Compared with healthy controls, fecal microbial diversities, as estimated by the Shannon index (B), Simpson index (C) and Invsimpson index (D), were no differences with HCC patients (all p > 0.05, Wilcoxon rank sum test). (E) A Venn diagram showed that 729 of the total 1002 OTUs were shared, notably, 148 of 1002 OTUs were unique for HCC. (F) Beta diversity was calculated using Bray-Curtis by NMDS. The red dot represents HCC Group, and the blue dot represents Healthy Control. HCC, hepatocellular carcinoma; OTU, Operational Taxonomy Unit; NMDS, Nonmetric multidimensional scaling. Fig. S4. No differences in gut microbes between patients with Cirrhotic HCC (n = 91) and Non-Cirrhotic HCC (n = 22), between patients with portal hypertension (n = 66) and without portal hypertension (n = 47), between patients with low-albumin (n = 16) and normal-albumin (n = 97). (A) The microbial community at genus level in patients with Cirrhotic HCC versus Non-Cirrhotic HCC. (B) The distributions of Bacteroides, Lachnospiracea incertae sedis and Clostridium XIVa normalized by Z- score among healthy controls, patients with Cirrhotic HCC and Non-Cirrhotic HCC. (C) No differences in Bacteroides, Lachnospiracea incertae sedis and Clostridium XIVa between patients with portal hypertension and without portal hypertension. (D) No differences in Bacteroides, Lachnospiracea incertae sedis and Clostridium XIVa between patients with low-albumin and normal-albumin. (Wilcoxon rank sum test) HCC, hepatocellular carcinoma. Fig. S5. The specific characterization of fecal microbiota to distinguish toxigenic types was analyzed by linear discriminant analysis (LDA) effect size (LEfSe) method. (A) LEfSe method identified the most differentially abundant taxons between healthy controls and HCC patients. The HCC-enriched taxa were indicated with a negative LDA score (red), and health-enriched taxa presented a positive score (green). Bacteroides, Lachnospiracea incertae sedis and Clostridium XIVa were significantly overrepresented in the feces of HCC patients. (B) LEfSe method identified the most differentially abundant taxons between Small HCC and Non-Small HCC. The Non-Small HCC-enriched taxa were indicated with a negative LDA score (red), and Small-HCC-enriched taxa presented a positive score (green). Bacteroides, Lachnospiracea incertae sedis and Clostridium XIVa were significantly overrepresented in the feces of patients in Non-Small HCC subgroup. log10(LDA score) = 3 as cut-off value. HCC, hepatocellular carcinoma. Fig. S6. Scatter diagrams for all 31 OTU-gene pairs. The x axis indicated the OTU abundance. The y axis indicated log2FC of gene expression calculated by GFOLD. Each point represented a patient (red: Non-Small HCC; blue: Small HCC) and some points coincided at the origin. Fig. S7. The expressions of 29 OTU-related genes in 23 Non-Small HCC (A) and 9 Small HCC patients (B, C). Parameter of edgeR: biological coefficient of variation (BCV) for 23 Non-Small HCC patients was 0.6090678 (D) and BCV for 9 Small HCC patients was 0.5872324 (E). Fig. S8. Overall survival and disease free survival for all 29 OTU-related genes based on GEPIA. Overall survival and disease free survival were identified by Log-rank test, a.k.a. the Mantel-Cox test (Cutoff-High and Cutoff-Low was both 50%). TRBC1 (official symbol) is named TRBV25–1 in GEPIA database. Fig. S9. Scatter diagrams for all 29 microbe-associated genes and levels of ALT/AST. The x axis indicated the levels of ALT/AST. The y axis indicated log2FC of gene expression calculated by GFOLD. Each point represented a patient (blue: ALT; red: AST). Fig. S10. Expressions of OTU-related genes for each cell type based on SMART-seq2 data ( http://cancer-pku.cn:3838/HCC ). Uniform Manifold Approximation and Projection (UMAP) plots showed 38 clusters identified by integrated analysis, colored by cell cluster (first plot). UMAP plots showed the cell distributions of tumor and adjacent liver from 6 patients (second plot). CARMIL2 was not found in this database. TRBC1 (official symbol) is named TRBV25–1 in this database. Fig. S11. The composition of stool color. Most of stool samples presented yellow, and showed no significant difference among the different cohorts. (A) The composition of stool color between healthy controls and HCC patients. (B) The composition of stool color in patients with Small HCC versus Non-Small HCC. HCC, hepatocellular carcinoma. Fig. S12. The abundance and distribution of stool moisture among the different cohorts by lyophilization assay on the frozen homogenized fecal material. Stool moisture content was determined in duplicate on the frozen homogenized fecal material (− 80 °C) as the percentage of stool mass loss after lyophilization. (A) The abundance of stool moisture between healthy controls and HCC patients. (B) The abundance of stool moisture in patients with Small HCC versus Non-Small HCC. (student’s t test) HCC, hepatocellular carcinoma.

Cancer stem cells (CSCs) are cells possessing abilities of self-renewal, differentiation, and tumorigenicity in NOD/SCID mice. Based on this definition, multiple cell surface markers (such as CD24, CD133, CD90, and EpCAM) as well as chemical methods are discovered to enrich liver CSCs in the recent decade. Accumulated studies have revealed molecular signatures and signaling pathways involved in regulating different liver CSCs. Among liver CSCs positive for different markers, some molecular features and regulatory pathways are commonly shared, while some are only unique in certain CSC populations. These studies imply that liver CSCs exhibit diverse heterogeneity, while a functional relationship also exists. The aim of this review is to revisit the society of liver CSCs and summarize the common or unique molecular features of known liver CSCs. We hope to call for attention of researchers on the relationship of the liver CSC subgroups and to provide clues on the hierarchical structure of the liver CSC society.

Additional file 3. Supplementary Methods (including detailed custom code and mathematical algorithm).

Liver cancers, which are mostly hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), are very aggressive tumors with poor prognosis. Therapeutic options with curative intent are largely limited to surgery and available systemic therapies show limited benefit. Signal transducer and activator of transcription 1 (STAT1) and 3 (STAT3) are key transcription factors activated by pro-inflammatory cytokines such as interferon-γ (IFN-γ) and interleukin-6 (IL-6). In this study, we combined in vitro cell culture experiments and immunohistochemical analyses of human HCC (N = 124) and CCA (N = 138) specimens. We observed that in the absence of STAT3, IL-6 induced the activation of STAT1 and its target genes suggesting that IL-6 derived from the tumor microenvironment may activate both STAT1 and STAT3 target genes in HCC tumor cells. In addition, STAT1 and STAT3 were highly activated in a subset of HCC, which exhibited a high degree of infiltrating CD8- and FOXP3-positive immune cells and PD-L1 expression. Our results demonstrate that STAT1 and STAT3 are expressed and activated in HCC and tumor infiltrating immune cells. In addition, HCC cases with high STAT1 and STAT3 expression also exhibited a high degree of immune cell infiltration, suggesting increased immunological tolerance.

Intronic miRNAs, residing in intronic regions of host genes, are thought to be co-transcribed from their host genes and present consistent expression patterns with host genes. Recent studies reported a few intronic miRNAs with discordant expression with their host genes. We therefore aimed to understand the expression pattern of intronic miRNAs and their host genes in hepatocellular carcinoma (HCC) and reveal possible associated molecular mechanisms. Our genome wide integration analysis of miRNA and mRNA transcriptomes, in three dataset from 550 patients with HCC, found that a large amount of miRNA-host gene pairs were discordantly expressed. Consistent results were also revealed in 775 breast cancer patients. Further, most of HCC-related intronic miRNAs were predicted to have distinct upstream regulators and independent proximal promoter signals from host genes. The discordant expression of representative pairs, miR-26s/CTDSPs, was validated experimentally. We have also identified the independent transcriptional start site, promoter signal, and transcriptional factor of miR-26b from its host gene. Collectively, discordant expression of intronic miRNAs and their host genes was relatively ubiquitous and the intronic miRNA "independent transcription" may partially contribute to such a phenotype.

Distal cholangiocarcinoma (dCCA) is a biliary tract cancer with a dismal prognosis and is often preceded by biliary intraepithelial neoplasia (BilIN), representing the most common biliary non-invasive precursor lesion. BilIN are histologically well defined but have not so far been characterised systematically at the molecular level. The aim of this study was to determine miRNA-regulated genes in cholangiocarcinogenesis via BilIN. We used a clinicopathologically well-characterised cohort of 12 dCCA patients. Matched samples of non-neoplastic biliary epithelia, BilIN and invasive tumour epithelia of each patient were isolated from formalin-fixed paraffin-embedded tissue sections by laser microdissection. The resulting 36 samples were subjected to total RNA extraction and the expression of 798 miRNAs was assessed using the Nanostring® technology. Candidate miRNAs were validated by RT-qPCR and functionally investigated following lentiviral overexpression in dCCA-derived cell lines. Potential direct miRNA target genes were identified by microarray and prediction algorithms and were confirmed by luciferase assay. We identified 49 deregulated miRNAs comparing non-neoplastic and tumour tissue. Clustering of these miRNAs corresponded to the three stages of cholangiocarcinogenesis, supporting the concept of BilIN as a tumour precursor. Two downregulated miRNAs, i.e. miR-451a (-10.9-fold down) and miR-144-3p (-6.3-fold down), stood out by relative decrease. Functional analyses of these candidates revealed a migration inhibitory effect in dCCA cell lines. Activating transcription factor 2 (ATF2) and A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) were identified as direct miR-451a target genes. Specific ATF2 inhibition by pooled siRNAs reproduced the inhibitory impact of miR-451a on cancer cell migration. Thus, our data support the concept of BilIN as a direct precursor of invasive dCCA at the molecular level. In addition, we identified miR-451a and miR-144-3p as putative tumour suppressors attenuating cell migration by inhibiting ATF2 in the process of dCCA tumorigenesis. © The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Ras mutations are present in only a subset of sporadic human cutaneous squamous cell carcinomas (cSCC) even though Ras is activated in most. This suggests that other mechanisms of Ras activation play a role in the disease. The aberrant expression of RasGRP1, a guanyl nucleotide exchange factor for Ras, is critical for mouse cSCC development through its ability to increase Ras activity. However, the role of RasGRP1 in human keratinocyte carcinogenesis remains unknown. Here we report that RasGRP1 is significantly elevated in human cSCC and that high RasGRP1 expression in human primary keratinocytes triggered activation of endogenous Ras and significant morphological changes including cytoplasmic vacuole formation and growth arrest. Moreover, RasGRP1-expressing cells were autophagic as indicated by LC3-II increase and the formation of LC3 punctae. In an in vitro organotypic skin model, wild type keratinocytes generated a well-stratified epithelium, while RasGRP1-expressing cells failed to do so. Finally, RasGRP1 induced transformation-like changes in skin cells from Li-Fraumeni patients with inactivating p53 mutations, demonstrating the oncogenic potential of this protein. These results support a role for RasGRP1 in human epidermal keratinocyte carcinogenesis and might serve as an important new therapeutic target.

Various cancer stem cell (CSC) biomarkers have been identified for hepatocellular carcinoma (HCC), but little is known about the implications of heterogeneity and shared molecular networks within the CSC population. Through miRNA profile analysis in an HCC cohort (n = 241) for five groups of CSC+ HCC tissues, i.e., EpCAM+, CD90+, CD133+, CD44+, and CD24+ HCC, we identified a 14-miRNA signature commonly altered among these five groups of CSC+ HCC. miR-192-5p, the top-ranked CSC miRNA, was liver-abundant and -specific and markedly downregulated in all five groups of CSC+ HCC from two independent cohorts (n = 613). Suppressing miR-192-5p in HCC cells significantly increased multiple CSC populations and CSC-related features through targeting PABPC4. Both TP53 mutation and hypermethylation of the mir-192 promoter impeded transcriptional activation of miR-192-5p in HCC cell lines and primary CSC+ HCC. This study reveals the circuit from hypermethylation of the mir-192 promoter through the increase in PABPC4 as a shared genetic regulatory pathway in various groups of primary CSC+ HCC. This circuit may be the driver that steers liver cells toward hepatic CSC cells, leading to hepatic carcinogenesis. Significance: miR-192-5p and its regulatory pathway is significantly abolished in multiple groups of HCC expressing high levels of CSC markers, which may represent a key event for hepatic carcinogenesis.

Human pluripotent stem cells (hPSCs) have potential applications in biological studies and regenerative medicine. However, precise genome editing in hPSCs remains time-consuming and labor-intensive. Here we demonstrate that the recently identified CRISPR-Cpf1 can be used to efficiently generate knockout and knockin hPSC lines. The unique properties of CRISPR-Cpf1, including shorter crRNA length and low off-target activity, are very attractive for many applications. In particular, we develop an unbiased drug-selection-based platform feasible for high-throughput screening in hPSCs and this screening system enables us to identify small molecules VE-822 and AZD-7762 that can promote CRISPR-Cpf1-mediated precise genome editing. Significantly, the combination of CRISPR-Cpf1 and small molecules provides a simple and efficient strategy for precise genome engineering., Precise genome editing in human pluripotent stem cells requires the development of methods for rapid and efficient genetic manipulation. Here, the authors screen for small molecules that enhance CRISPR-Cpf1-mediated genome engineering.

UNLABELLED The cell fate determinant Numb is aberrantly expressed in cancer. Numb is alternatively spliced, with one isoform containing a long proline-rich region (PRR(L) ) compared to the other with a short PRR (PRR(S) ). Recently, PRR(L) was reported to enhance proliferation of breast and lung cancer cells. However, the importance of Numb alternative splicing in hepatocellular carcinoma (HCC) remains unexplored. We report here that Numb PRR(L) expression is increased in HCC and associated with early recurrence and reduced overall survival after surgery. In a panel of HCC cell lines, PRR(L) generally promotes and PRR(S) suppresses proliferation, migration, invasion, and colony formation. Knockdown of PRR(S) leads to increased Akt phosphorylation and c-Myc expression, and Akt inhibition or c-Myc silencing dampens the proliferative impact of Numb PRR(S) knockdown. In the cell models explored in this study, alternative splicing of Numb PRR isoforms is coordinately regulated by the splicing factor RNA-binding Fox domain containing 2 (RbFox2) and the kinase serine/arginine protein-specific kinase 2 (SRPK2). Knockdown of the former causes accumulation of PRR(L) , while SRPK2 knockdown causes accumulation of PRR(S) . The subcellular location of SRPK2 is regulated by the molecular chaperone heat shock protein 90, and heat shock protein 90 inhibition or knockdown phenocopies SRPK2 knockdown in promoting accumulation of Numb PRR(S) . Finally, HCC cell lines that predominantly express PRR(L) are differentially sensitive to heat shock protein 90 inhibition. CONCLUSION Alternative splicing of Numb may provide a useful prognostic biomarker in HCC and is pharmacologically tractable.

BRCA1 is a well established tumor suppressor gene, which is involved in many cellular processes, including DNA damage repair, cell cycle control, apoptosis, as well as transcriptional control. In this work, we have found that BRCA1 is involved in regulating TGF-β1/Smad pathway. The loss of endogenous BRCA1 greatly attenuated TGF-β1-induced growth inhibition and cell cycle G1 arrest. BRCA1 greatly maintains stability of Smad4 protein, and the loss of BRCA1 results in Smad4 down-regulation, which is likely related to its downstream gene Gadd45a. Gadd45a is able to interact with β-Trcp1, a-F-box protein of SCF E3 ligase, and consequently suppresses the ubiquitin-degradation of Smad4 by SCF(β-trcp1), as reflected by the observations that the induction of Gadd45a substantially stabilizes Smad4 protein. In addition, exogenous expression of Gadd45a can largely rescue the protein level of Smad4 in BRCA1 deficient cells. These results further demonstrate that BRCA1 may act as an important negative regulator in cell cycle progression and tumorigenesis through regulating the stability of Smad4, and define a novel link that connects BRCA1 to TGF-β1/Smad pathway.

Deregulation of microRNAs (miRNAs) is a typical feature of human hepatocellular carcinoma (HCC). However, the in vivo relevance of miRNAs along hepatocarcinogenesis remains largely unknown. Here, we show that liver tumors induced in mice by c-Myc overexpression or AKT/Ras co-expression exhibit distinct miRNA expression profiles. Among the downregulated miRNAs, eight (miR-101, miR-107, miR-122, miR-29, miR-365, miR-375, miR-378, and miR-802) were selected and their tumor suppressor activity was determined by overexpressing each of them together with c-Myc or AKT/Ras oncogenes in mouse livers via hydrodynamic transfection. The tumor suppressor activity of these microRNAs was extremely heterogeneous in c-Myc and AKT/Ras mice: while miR-378 had no tumor suppressor activity, miR-107, mir-122, miR-29, miR-365 and miR-802 exhibited weak to moderate tumor suppressor potential. Noticeably, miR-375 showed limited antineoplastic activity against c-Myc driven tumorigenesis, whereas it strongly inhibited AKT/Ras induced hepatocarcinogenesis. Furthermore, miR-101 significantly suppressed both c-Myc and AKT/Ras liver tumor development. Altogether, the present data demonstrate that different oncogenes induce distinct miRNA patterns, whose modulation differently affects hepatocarcinogenesis depending on the driving oncogenes. Finally, our findings support a strong tumor suppressor activity of miR-101 in liver cancer models regardless of the driver oncogenes involved, thus representing a promising therapeutic target in human HCC.

Ras is frequently activated in cutaneous squamous cell carcinoma, a prevalent form of skin cancer. However, the pathways that contribute to Ras-induced transformation have not been entirely elucidated. We have previously demonstrated that in transgenic mice, overexpression of the Ras activator RasGRP1 promotes the formation of spontaneous skin tumors and enhances malignant progression in the multistage carcinogenesis skin model that relies on the oncogenic activation of H-Ras. Utilizing a RasGRP1 knockout mouse model (RasGRP1 KO), we now show that lack of RasGRP1 reduced the susceptibility to skin tumorigenesis. The dependency on RasGRP1 was associated with a diminished response to the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Specifically, we found impairment of epidermal hyperplasia induced by TPA through keratinocyte proliferation. Using a keratinocyte cell line that carries a ras oncogenic mutation, we also demonstrated that RasGRP1 could further activate Ras in response to TPA. Thus, we propose that RasGRP1 upregulates signaling from Ras and contributes to epidermal tumorigenesis by increasing the total dosage of active Ras.

Nucleophosmin (NPM) is a ubiquitously expressed phosphoprotein involved in many cellular processes. Phosphorylation is considered the major regulatory mechanism of the NPM protein, associated with diverse cellular events. In this study, we characterized the phosphorylation status of several physiological phosphorylation sites of NPM, especially the newly confirmed in vivo site threonine 95 (Thr95). NPM-Thr95 exhibits a transient and cell cycle-dependent phosphorylation state compared to several other in vivo phosphorylation sites examined, including Ser4, Thr199 and Thr234/Thr237. In addition, we characterized a functional interaction between NPM and the peptidyl-prolyl isomerase Pin1, which specifically bind to each other during mitosis. The demonstration of this binding represents a novel post-phosphorylation regulatory mechanism for NPM that has not been investigated before. Mutated Pin1 putative binding sites result in defected cell division and reduced number of mitotic cells, suggesting that post-phosphorylation is important for NPM in regulating cell cycle progression.

Intrahepatic cholangiocellular carcinoma (ICC) is the second most common type of primary liver cancer. However, its tumor heterogeneity and molecular characteristics are largely unknown. In this study, we conducted transcriptomic profiling of 23 ICC and combined hepatocellular cholangiocarcinoma tumor specimens from Asian patients using Affymetrix messenger RNA (mRNA) and NanoString microRNA microarrays to search for unique gene signatures linked to tumor subtypes and patient prognosis. We validated the signatures in an additional 68 ICC cases derived from Caucasian patients. We found that both mRNA and microRNA expression profiles could independently classify Asian ICC cases into two main subgroups, one of which shared gene expression signatures with previously identified hepatocellular carcinoma (HCC) with stem cell gene expression traits. ICC-specific gene signatures could predict survival in Asian HCC cases and independently in Caucasian ICC cases. Integrative analyses of the ICC-specific mRNA and microRNA expression profiles revealed that a common signaling pathway linking miR-200c signaling to epithelial-mesenchymal transition (EMT) was preferentially activated in ICC with stem cell gene expression traits. Inactivation of miR-200c resulted in an induction of EMT, whereas activation of miR-200c led to a reduction of EMT including a reduced cell migration and invasion in ICC cells. We also found that miR-200c and neural cell adhesion molecule 1 (NCAM1) expression were negatively correlated and their expression levels were predictive of survival in ICC samples. NCAM1, a known hepatic stem/progenitor cell marker, was experimentally demonstrated to be a direct target of miR-200c. Conclusion: Our results indicate that ICC and HCC share common stem-like molecular characteristics and poor prognosis. We suggest that the specific components of EMT may be exploited as critical biomarkers and clinically relevant therapeutic targets for an aggressive form of stem cell-like ICC. (HEPATOLOGY 2012;56:1792–1803)

Solid tumors are thought to contain cancer stem cells (CSCs) as a distinct population responsible for tumor relapse and metastasis due to their abilities to self-renew, differentiate, and give rise to a new tumor in local or distant organs. CSCs have been identified in many tumor types, including hepatocellular carcinoma (HCC), the fifth most common and third most deadly malignancy with observable heterogeneity. Numerous studies have shown that hepatic CSCs could be enriched via different cell surface markers, eg, CD13, CD24, CD44, CD90, CD133, EpCAM (CD326), and OV6. They also could be identified through functional assays such as isolating the side population cells by Hoechst dye staining or screening cells with a high activity of aldehyde dehydrogenase. Functional characterization of hepatic CSCs has revealed several deregulated signaling pathways, such as Wnt/β-catenin, AKT, transforming growth factor-beta (TGF-β), interleukin (IL)-6/STAT3 pathways to be critical in inducing "stemness" of HCC and in promoting self-renewal, tumorigenicity, and chemoresistance. An increased understanding of hepatic CSC biology facilitated the development of new diagnostic, prognostic, and therapeutic strategies for improving HCC clinical management. In this review, we summarize recent evidence including the identification of the hepatic CSC and its underlying biological mechanisms, and discuss the potential clinical implications in HCC.

BACKGROUND: Hepatocellular carcinoma is a common and aggressive cancer that occurs mainly in men. We examined microRNA expression patterns, survival, and response to interferon alfa in both men and women with the disease. METHODS: We analyzed three independent cohorts that included a total of 455 patients with hepatocellular carcinoma who had undergone radical tumor resection between 1999 and 2003. MicroRNA-expression profiling was performed in a cohort of 241 patients with hepatocellular carcinoma to identify tumor-related microRNAs and determine their association with survival in men and women. In addition, to validate our findings, we used quantitative reverse-transcriptase-polymerase- chain-reaction assays to measure microRNAs and assess their association with survival and response to therapy with interferon alfa in 214 patients from two independent, prospective, randomized, controlled trials of adjuvant interferon therapy. RESULTS: In patients with hepatocellular carcinoma, the expression of miR-26a and miR-26b in nontumor liver tissue was higher in women than in men. Tumors had reduced levels of miR-26 expression, as compared with paired noncancerous tissues, which indicated that the level of miR-26 expression was also associated with hepatocellular carcinoma. Moreover, tumors with reduced miR-26 expression had a distinct transcriptomic pattern, and analyses of gene networks revealed that activation of signaling pathways between nuclear factor κB and interleukin-6 might play a role in tumor development. Patients whose tumors had low miR-26 expression had shorter overall survival but a better response to interferon therapy than did patients whose tumors had high expression of the microRNA. CONCLUSIONS: The expression patterns of microRNAs in liver tissue differ between men and women with hepatocellular carcinoma. The miR-26 expression status of such patients is associated with survival and response to adjuvant therapy with interferon alfa. Copyright © 2009 Massachusetts Medical Society. All rights reserved., published_or_final_version

UNLABELLED Therapies that target cancer stem cells (CSCs) hold promise in eliminating cancer burden. However, normal stem cells are likely to be targeted owing to their similarities to CSCs. It is established that epithelial cell adhesion molecule (EpCAM) is a biomarker for normal hepatic stem cells (HpSCs), and EpCAM(+) AFP(+) hepatocellular carcinoma (HCC) cells have enriched hepatic CSCs. We sought to determine whether specific microRNAs (miRNAs) exist in hepatic CSCs that are not expressed in normal HpSCs. We performed a pair-wise comparison of the miRNA transcriptome of EpCAM(+) and corresponding EpCAM(-) cells isolated from two primary HCC specimens, as well as from two fetal livers and three healthy adult liver donors by small RNA deep sequencing. We found that miR-150, miR-155, and miR-223 were preferentially highly expressed in EpCAM(+) HCC cells, which was further validated. Their gene surrogates, identified using miRNA and messenger RNA profiling in a cohort of 292 HCC patients, were associated with patient prognosis. We further demonstrated that miR-155 was highly expressed in EpCAM(+) HCC cells, compared to corresponding EpCAM(-) HCC cells, fetal livers with enriched normal hepatic progenitors, and normal adult livers with enriched mature hepatocytes. Suppressing miR-155 resulted in a decreased EpCAM(+) fraction in HCC cells and reduced HCC cell colony formation, migration, and invasion in vitro. The reduced levels of identified miR-155 targets predicted the shortened overall survival and time to recurrence of HCC patients. CONCLUSION miR-155 is highly elevated in EpCAM(+) HCC cells and might serve as a molecular target to eradicate the EpCAM(+) CSC population in human HCCs.

Globally, hepatocellular carcinoma (HCC) accounts for 70%-85% of primary liver cancers and ranks as the second leading cause of male cancer death. Serum alpha-fetoprotein (AFP), normally highly expressed in the liver only during fetal development, is reactivated in 60% of HCC tumors and associated with poor patient outcome. We hypothesize that AFP+ and AFP− tumors differ biologically. Multivariable analysis in 237 HCC cases demonstrates that AFP level predicts poor survival independent of tumor stage (P

Background & Aims: Adjuvant therapies for hepatocellular carcinoma (HCC) such as interferon-alpha are effective only in a subset of patients. Previously we found that HCC patients with low level of miR-26 have survival benefits from interferon-alpha. The purpose of this study is to develop a standardized miR-26 diagnostic test (referred as MIR26-DX) to assist identification of candidate HCC patients for adjuvant interferon-alpha therapy. Methods: We developed a multiplex reverse-transcription quantitative polymerase-chain-reaction assay to determine the levels of two HCC-related miR-26 transcripts along with six small RNA reference transcripts. We evaluated archived paraffin-embedded tissues from three cohorts of HCC patients (n=248) who underwent radical resection at three different clinical centers. Fifty-two percent of them underwent adjuvant interferon-alpha therapy. We used Cox-Mantel log-rank test to evaluate patient survival. Results: We found that the multiplexing assay was stable and reproducible regardless of differences in sample preparations and operators. We developed a matrix template and a scoring algorithm based on a training cohort (n=129) to assign HCC patients, and then applied the template in two test cohorts (n=119). The proportions of HCC patients assigned as low miR-26 by this algorithm were 68, 4, and 63 percent in the training cohort and two test cohorts, respectively. Consistently, HCC with low miR-26 had a favorable response to interferon-alpha with improved median overall survival (≥3year). Conclusions: MIR26-DX is a simple and reliable companion diagnostic test to select HCC patients for adjuvant interferon-alpha therapy.

SummaryMembers of sirtuin family regulate multiple critical biological processes, yet their role in carcinogenesis remains controversial. To investigate the physiological functions of SIRT2 in development and tumorigenesis, we disrupted Sirt2 in mice. We demonstrated that SIRT2 regulates the anaphase-promoting complex/cyclosome activity through deacetylation of its coactivators, APCCDH1 and CDC20. SIRT2 deficiency caused increased levels of mitotic regulators, including Aurora-A and -B that direct centrosome amplification, aneuploidy, and mitotic cell death. Sirt2-deficient mice develop gender-specific tumorigenesis, with females primarily developing mammary tumors, and males developing more hepatocellular carcinoma (HCC). Human breast cancers and HCC samples exhibited reduced SIRT2 levels compared with normal tissues. These data demonstrate that SIRT2 is a tumor suppressor through its role in regulating mitosis and genome integrity.