Single tumor-initiating cells evade immune clearance by recruiting type II macrophages.

Tumor infiltrated type II (M2) macrophages promote tumorigenesis by suppressing immune clearance, promoting proliferation, and stimulating angiogenesis. Interestingly, macrophages were also found to enrich in small foci of altered hepatocytes containing liver tumor-initiating cells (TICs). However, whether and how TICs specifically recruit macrophages and the function of these macrophages in tumor initiation remain unknown due to technical difficulties. In this study, by generating genetically defined liver TICs, we demonstrate that TICs actively recruit M2 macrophages from as early as the single-cell stage. Elimination of TIC-associated macrophages (TICAMs) abolishes tumorigenesis in a manner dependent on the immune system. Mechanistically, activation of the Hippo pathway effector Yes-associated protein (YAP) underlies macrophage recruitment by TICs. These results demonstrate for the first time that macrophages play a decisive role in the survival of single TICs in vivo and provide a proof of principle for TIC elimination by targeting YAP or M2 macrophages.

Blocking hepatocarcinogenesis by a cytochrome P450 family member with female-preferential expression.

OBJECTS: The incidence of hepatocellular carcinoma (HCC) shows an obvious male dominance in rodents and humans. We aimed to identify the key autosomal liver-specific sex-related genes and investigate their roles in hepatocarcinogenesis. DESIGN: Two HCC cohorts (n=551) with available transcriptome and metabolome data were used. Class comparisons of omics data and ingenuity pathway analysis were performed to explore sex-related molecules and their associated functions. Functional assays were employed to investigate roles of the key candidates, including cellular assays, molecular assays and multiple orthotopic HCC mouse models. RESULTS: A global comparison of multiple omics data revealed 861 sex-related molecules in non-tumour liver tissues between female and male HCC patients, which denoted a significant suppression of cancer-related diseases and functions in female liver than male. A member of cytochrome P450 family, CYP39A1, was one of the top liver-specific candidates with significantly higher levels in female vs male liver. In HCC tumours, CYP39A1 expression was dramatically reduced in over 90% HCC patients. Exogenous CYP39A1 significantly blocked tumour formation in both female and male mice and partially reduced the sex disparity of hepatocarcinogenesis. The HCC suppressor role of CYP39A1 did not rely on its known P450 enzyme activity but its C-terminal region, by which CYP39A1 impeded the transcriptional activation activity of c-Myc, leading to a significant inhibition of hepatocarcinogenesis. CONCLUSIONS: The liver-specific CYP39A1 with female-preferential expression was a strong suppressor of HCC development. Strategies to up-regulate CYP39A1 might be promising methods for HCC treatment in both women and men in future.

MiR-125b Loss Activated HIF1α/pAKT Loop, Leading to Transarterial Chemoembolization Resistance in Hepatocellular Carcinoma.

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 ß (PDGFß)/pAKT/HIF1α loop of HIF1α translation by targeting the PDGFß 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.

miRNA profiling of biliary intraepithelial neoplasia reveals stepwise tumorigenesis in distal cholangiocarcinoma via the miR-451a/ATF2 axis.

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.

Liver cancer stem cells as a hierarchical society: yes or no?

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.

Identification of microRNAs specific for epithelial cell adhesion molecule-positive tumor cells in hepatocellular carcinoma.

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.

Alternative splicing of the cell fate determinant Numb in hepatocellular carcinoma.

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.

Modulation of miR-29 expression by α-fetoprotein is linked to the hepatocellular carcinoma epigenome.

UNLABELLED: 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<0.043). Using microarray-based global microRNA (miRNA) profiling, we found that miRNA-29 (miR-29) family members were the most significantly (P<0.001) down-regulated miRNAs in AFP+ tumors. Consistent with miR-29's role in targeting DNA methyltransferase 3A (DNMT3A), a key enzyme regulating DNA methylation, we found a significant inverse correlation (P<0.001) between miR-29 and DNMT3A gene expression, suggesting that they might be functionally antagonistic. Moreover, global DNA methylation profiling reveals that AFP+ and AFP- HCC tumors have distinct global DNA methylation patterns and that increased DNA methylation is associated with AFP+ HCC. Experimentally, we found that AFP expression in AFP- HCC cells induces cell proliferation, migration, and invasion. Overexpression of AFP, or conditioned media from AFP+ cells, inhibits miR-29a expression and induces DNMT3A expression in AFP- HCC cells. AFP also inhibited transcription of the miR-29a/b-1 locus, and this effect is mediated through c-MYC binding to the transcript of miR-29a/b-1. Furthermore, AFP expression promotes tumor growth of AFP- HCC cells in nude mice. CONCLUSION: Tumor biology differs considerably between AFP+ HCC and AFP- HCC; AFP is a functional antagonist of miR-29, which may contribute to global epigenetic alterations and poor prognosis in HCC.

Targeted deletion of RasGRP1 impairs skin tumorigenesis.

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.

Integrated metabolite and gene expression profiles identify lipid biomarkers associated with progression of hepatocellular carcinoma and patient outcomes.

BACKGROUND & AIMS: We combined gene expression and metabolic profiling analyses to identify factors associated with outcomes of patients with hepatocellular carcinoma (HCC). METHODS: We compared metabolic and gene expression patterns between paired tumor and nontumor tissues from 30 patients with HCC, and validated the results using samples from 356 patients with HCC. A total of 469 metabolites were measured using liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry. Metabolic and genomic data were integrated, and Kaplan-Meier and Cox proportional hazards analyses were used to associate specific patterns with patient outcomes. Associated factors were evaluated for their effects on cancer cells in vitro and tumor formation in nude mice. RESULTS: We identified 28 metabolites and 169 genes associated with aggressive HCC. Lipid metabolites of stearoyl-CoA-desaturase (SCD) activity were associated with aberrant palmitate signaling in aggressive HCC samples. Expression of gene products associated with these metabolites, including SCD, were associated independently with survival times and tumor recurrence in the test and validation sets. Combined expression of SCD and α-fetoprotein were associated with outcomes of patients with early-stage HCC. Levels of monounsaturated palmitic acid, the product of SCD activity, were increased in aggressive HCCs; monounsaturated palmitic acid increased migration and invasion of cultured HCC cells and colony formation by HCC cells. HCC cells that expressed small interfering RNA against SCD had decreased cell migration and colony formation in culture and reduced tumorigenicity in mice. CONCLUSIONS: By using a combination of gene expression and metabolic profile analysis, we identified a lipogenic network that involves SCD and palmitate signaling and was associated with HCC progression and patient outcomes. The microarray platform and data have been submitted to the Gene Expression Omnibus public database at NCBI following MIAME guidelines. Accession numbers: GPL4700 (platform), and GSE6857 (samples).

The HRD1-SEL1L ubiquitin ligase regulates stress granule homeostasis in couple with distinctive signaling branches of ER stress.

Stress granules (SGs) are membrane-less cellular compartments which are dynamically assembled via biomolecular condensation mechanism when eukaryotic cells encounter environmental stresses. SGs are important for gene expression and cell fate regulation. Dysregulation of SG homeostasis has been linked to human neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we report that the HRD1-SEL1L ubiquitin ligase complex specifically regulates the homeostasis of heat shock-induced SGs through the ubiquitin-proteasome system (UPS) and the UPS-associated ATPase p97. Mechanistically, the HRD1-SEL1L complex mediates SG homeostasis through the BiP-coupled PERK-eIF2α signaling axis of endoplasmic reticulum (ER) stress, thereby coordinating the unfolded protein response (UPR) with SG dynamics. Furthermore, we show that the distinctive branches of ER stress play differential roles in SG homeostasis. Our study indicates that the UPS and the UPR together via the HRD1-SEL1L ubiquitin ligase to maintain SG homeostasis in a stressor-dependent manner.

Smad4 sequestered in SFPQ condensates prevents TGF-ß tumor-suppressive signaling.

Loss of TGF-ß growth-inhibitory responses is a hallmark of human cancer. However, the molecular mechanisms underlying the TGF-ß resistance of cancer cells remain to be fully elucidated. Splicing factor proline- and glutamine-rich (SFPQ) is a prion-like RNA-binding protein that is frequently upregulated in human cancers. In this study, we identified SFPQ as a potent suppressor of TGF-ß signaling. The ability of SFPQ to suppress TGF-ß responses depends on its prion-like domain (PrLD) that drives liquid-liquid phase separation (LLPS). Mechanistically, SFPQ physically restrained Smad4 in its condensates, which excluded Smad4 from the Smad complex and chromatin occupancy and thus functionally dampened Smad-dependent transcriptional responses. Accordingly, SFPQ deficiency or loss of phase separation activities rendered human cells hypersensitive to TGF-ß responses. Together, our data identify an important function of SFPQ through LLPS that suppresses Smad transcriptional activation and TGF-ß tumor-suppressive activity.

Sexually Dimorphic Response to Hepatic Injury in Newborn Suffering from Intrauterine Growth Restriction.

Intrauterine growth restriction (IUGR), when a fetus does not grow as expected, is associated with a reduction in hepatic functionality and a higher risk for chronic liver disease in adulthood. Utilizing early developmental plasticity to reverse the outcome of poor fetal programming remains an unexplored area. Focusing on the biochemical profiles of neonates and previous transcriptome findings, piglets from the same fetus are selected as models for studying IUGR. The cellular landscape of the liver is created by scRNA-seq to reveal sex-dependent patterns in IUGR-induced hepatic injury. One week after birth, IUGR piglets experience hypoxic stress. IUGR females exhibit fibroblast-driven T cell conversion into an immune-adapted phenotype, which effectively alleviates inflammation and fosters hepatic regeneration. In contrast, males experience even more severe hepatic injury. Prolonged inflammation due to disrupted lipid metabolism hinders intercellular communication among non-immune cells, which ultimately impairs liver regeneration even into adulthood. Additionally, Apolipoprotein A4 (APOA4) is explored as a novel biomarker by reducing hepatic triglyceride deposition as a protective response against hypoxia in IUGR males. PPARα activation can mitigate hepatic damage and meanwhile restore over-expressed APOA4 to normal in IUGR males. The pioneering study offers valuable insights into the sexually dimorphic responses to hepatic injury during IUGR.

Oncogenic Roles of Laminin Subunit Gamma-2 in Intrahepatic Cholangiocarcinoma via Promoting EGFR Translation.

Intrahepatic cholangiocarcinoma (iCCA) is a highly lethal biliary epithelial cancer in the liver. Here, Laminin subunit gamma-2 (LAMC2) with important oncogenic roles in iCCA is discovered. In a total of 231 cholangiocarcinoma patients (82% of iCCA patients) across four independent cohorts, LAMC2 is significantly more abundant in iCCA tumor tissue compared to normal bile duct and non-tumor liver. Among 26.3% of iCCA patients, LAMC2 gene is amplified, contributing to its over-expression. Functionally, silencing LAMC2 significantly blocks tumor formation in orthotopic iCCA mouse models. Mechanistically, it promotes EGFR protein translation via interacting with nascent unglycosylated EGFR in the endoplasmic reticulum (ER), resulting in activated EGFR signaling. LAMC2-mediated EGFR translation also depends on its interaction with the ER chaperone BiP via their C-terminus. Together LAMC2 and BiP generate a binding "pocket" of nascent EGFR and facilitate EGFR translation. Consistently, LAMC2-high iCCA patients have poor prognosis in two iCCA cohorts. LAMC2-high iCCA cells are highly sensitive to EGFR tyrosine kinase inhibitors (TKIs) treatment both in vitro and in vivo. Together, these data demonstrate LAMC2 as an oncogenic player in iCCA by promoting EGFR translation and an indicator to identify iCCA patients who may benefit from available EGFR-targeted TKIs therapies.

The RNF214-TEAD-YAP signaling axis promotes hepatocellular carcinoma progression via TEAD ubiquitylation.

RNF214 is an understudied ubiquitin ligase with little knowledge of its biological functions or protein substrates. Here we show that the TEAD transcription factors in the Hippo pathway are substrates of RNF214. RNF214 induces non-proteolytic ubiquitylation at a conserved lysine residue of TEADs, enhances interactions between TEADs and YAP, and promotes transactivation of the downstream genes of the Hippo signaling. Moreover, YAP and TAZ could bind polyubiquitin chains, implying the underlying mechanisms by which RNF214 regulates the Hippo pathway. Furthermore, RNF214 is overexpressed in hepatocellular carcinoma (HCC) and inversely correlates with differentiation status and patient survival. Consistently, RNF214 promotes tumor cell proliferation, migration, and invasion, and HCC tumorigenesis in mice. Collectively, our data reveal RNF214 as a critical component in the Hippo pathway by forming a signaling axis of RNF214-TEAD-YAP and suggest that RNF214 is an oncogene of HCC and could be a potential drug target of HCC therapy.

Integrative genomic identification of genes on 8p associated with hepatocellular carcinoma progression and patient survival.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is an aggressive malignancy; its mechanisms of development and progression are poorly understood. We used an integrative approach to identify HCC driver genes, defined as genes whose copy numbers associate with gene expression and cancer progression. METHODS: We combined data from high-resolution, array-based comparative genomic hybridization and transcriptome analysis of HCC samples from 76 patients with hepatitis B virus infection with data on patient survival times. Candidate genes were functionally validated using in vitro and in vivo models. RESULTS: Unsupervised analyses of array comparative genomic hybridization data associated loss of chromosome 8p with poor outcome (reduced survival time); somatic copy number alterations correlated with expression of 27.3% of genes analyzed. We associated expression levels of 10 of these genes with patient survival times in 2 independent cohorts (comprising 319 cases of HCC with mixed etiology) and 3 breast cancer cohorts (637 cases). Among the 10-gene signature, a cluster of 6 genes on 8p, (DLC1, CCDC25, ELP3, PROSC, SH2D4A, and SORBS3) were deleted in HCCs from patients with poor outcomes. In vitro and in vivo analyses indicated that the products of PROSC, SH2D4A, and SORBS3 have tumor-suppressive activities, along with the known tumor suppressor gene DLC1. CONCLUSIONS: We used an unbiased approach to identify 10 genes associated with HCC progression. These might be used in assisting diagnosis and to stage tumors based on gene expression patterns.

Transcriptomic profiling reveals hepatic stem-like gene signatures and interplay of miR-200c and epithelial-mesenchymal transition in intrahepatic cholangiocarcinoma.

UNLABELLED: 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.

Chromosome 8p engineering reveals increased metastatic potential targetable by patient-specific synthetic lethality in liver cancer.

Large-scale chromosomal aberrations are prevalent in human cancer, but their function remains poorly understood. We established chromosome-engineered hepatocellular carcinoma cell lines using CRISPR-Cas9 genome editing. A 33-mega-base pair region on chromosome 8p (chr8p) was heterozygously deleted, mimicking a frequently observed chromosomal deletion. Using this isogenic model system, we delineated the functional consequences of chr8p loss and its impact on metastatic behavior and patient survival. We found that metastasis-associated genes on chr8p act in concert to induce an aggressive and invasive phenotype characteristic for chr8p-deleted tumors. Genome-wide CRISPR-Cas9 viability screening in isogenic chr8p-deleted cells served as a powerful tool to find previously unidentified synthetic lethal targets and vulnerabilities accompanying patient-specific chromosomal alterations. Using this target identification strategy, we showed that chr8p deletion sensitizes tumor cells to targeting of the reactive oxygen sanitizing enzyme Nudix hydrolase 17. Thus, chromosomal engineering allowed for the identification of novel synthetic lethalities specific to chr8p loss of heterozygosity.

MicroRNA expression, survival, and response to interferon in liver cancer.

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 kappaB 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.