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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

Loss of TGF-β tumour suppressive response is a hallmark of human cancers. As a central player in TGF-β signal transduction, SMAD4 (also known as DPC4) is frequently mutated or deleted in gastrointestinal and pancreatic cancer. However, such genetic alterations are rare in most cancer types and the underlying mechanism for TGF-β resistance is not understood. Here we describe a mechanism of TGF-β resistance in ALK-positive tumours, including lymphoma, lung cancer and neuroblastoma. We demonstrate that, in ALK-positive tumours, ALK directly phosphorylates SMAD4 at Tyr 95. Phosphorylated SMAD4 is unable to bind to DNA and fails to elicit TGF-β gene responses and tumour suppressing responses. Chemical or genetic interference of the oncogenic ALK restores TGF-β responses in ALK-positive tumour cells. These findings reveal that SMAD4 is tyrosine-phosphorylated by an oncogenic tyrosine kinase during tumorigenesis. This suggests a mechanism by which SMAD4 is inactivated in cancers and provides guidance for targeted therapies in ALK-positive cancers. Zhang et al. show that ALK phosphorylates SMAD4 at Tyr 95 to block its binding to DNA, representing a mutation-independent mechanism for blocking the tumour suppressor function of TGF-β in ALK-positive cancers.

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.

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.

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.

Background The incidence of hepatocellular cancer (HCC) is increasing, and we sought to characterize the differences and trends in HCC over 2 decades in Hawaii. Methods This retrospective study of 821 HCC cases analyzed risk factors, diabetes, alpha-fetoprotein (AFP), tumor characteristics, and treatment, comparing 5-year eras (1993 to 2012). Results With succeeding eras, there were fewer Asians, immigrants, and hepatitis B–related HCC. Hepatitis C, diabetes, hyperlipidemia, and body mass index have increased. Over time, more patients had normal AFP, and normal AFP was seen more often in nonviral HCC (49.6% vs 33.2%, P = .007). Over time, the proportion of patients who underwent resection or transplant was stable, but fewer patients underwent no therapy. Conclusions Characteristics of HCC are changing, and diagnosis may be more difficult as metabolic factors are becoming more important than viral factors. AFP seems to be a less important biomarker, and clearly, better diagnostic tools will be necessary to identify HCC in the future.

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.

Hepatocellular carcinoma (HCC), a major type of liver cancer, is a clinically and biologically heterogeneous malignancy. Recent advances in next-generation sequencing (NGS), a massively parallel nucleotide sequencing technology, allow researchers for cost-effective and simultaneous identification of genetic alterations, transcriptomic changes at much greater sensitivity, and accuracy. A large scale of mutational screening, gene expression examination, and their integration via NGS have been done in HCCs. These studies may enable the identification of new HCC genetic drivers and provide clues for HCC treatment using molecular-targeted therapies. The recent HCC NGS studies in this decade and their potential clinical utilization, with a focus on HCC heterogeneity, are discussed in this chapter.

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.

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

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.

Cancer Stem cells (CSCs) are the source of many solid tumor types including hepatocellular carcinoma. MicroRNAs (miRNAs) are small noncoding RNAs and have been showed to be associated with hepatic CSCs. Here, we described methods to screen hepatic CSC-related miRNAs, and to validate and examine their expressions and functions in vitro and in vivo, which contribute to the maintenance of stemness and differentiation of hepatic CSCs.

MicroRNAs are small noncoding RNAs that function to control gene expression. These small RNAs have been shown to contribute to the control of cell growth, differentiation and apoptosis, important features related to cancer development and progression. In fact, recent studies have shown the utility of microRNAs as cancer-related biomarkers. This is due to the finding that microRNAs display altered expression profiles in cancers versus normal tissue. In addition, microRNAs have been associated with cancer progression. In this review, the mechanisms to alter microRNA expression and their relation to cancer will be addressed. Moreover, the potential application of microRNAs in clinical settings will also be highlighted. Finally, the challenges regarding the translation of research involving microRNAs to the clinical realm will be discussed.

p53 mutations have been found in many esophageal squamous cell carcinoma (ESCC) clinical specimens and cell lines. We reasoned that functional inactivation of wild-type p53 or the functional activation of mutant-type p53 might exist in these specimens and cell lines. In this study, we identified the correlation between p53 functional activation and its genotype in five different ESCC cell lines. To examine the potential p53 activation in a certain ESCC cell line, DNA damage methods including X-ray exposure and cisplatin treatment were employed to treat cells. Further, the expression of p53 protein and four transcripts of well-known p53 target genes were investigated using Western blot and reverse transcriptionpolymerase chain reaction (RT-PCR) after cell exposure to DNA damage. The results showed that in KYSE 30 cell line with mutant p53 and KYSE 150 with wild-type p53, p53 could be activated by DNA damages. However, p53 could not be activated following the DNA damages in YES 2 with wild-type p53, KYSE 70 with mutant p53, and EC9706 with unknown p53 genotype. All our data indicated that p53 function in certain cells is not closely correlated with its genotype. To judge p53 function in a particular cell line, it is important to examine the p53 functional activation, but not to simply rely on the p53 genotype.

Hepatocellular carcinoma (HCC) is the fifth most common malignant cancer and the third leading cause of cancer death worldwide. Molecular profiling of changes in gene expression has improved our understanding of the HCC mechanism, allowing the identification of biomarkers for HCC diagnosis and HCC patient stratification for prognosis and therapy. Recently, a new group of molecules, microRNAs, has been discovered to be aberrantly expressed in HCC and some of them are functionally involved in HCC carcinogenesis and progression. Further, certain microRNAs are associated with HCC or related to HCC subtypes, implying the potential of microRNAs for HCC patient stratification of diagnosis and prognosis. Some of these HCC-associated microRNAs have been validated in independent cohorts, paving the way for developing clinically useful platforms to assess HCC risk, aiding HCC diagnosis and assisting in HCC patient stratification with the potential for personalized adjuvant therapy. Here, we mainly focus on the diagnostic and prognostic roles of miRNAs as a group of new biomarkers for HCC.

MicroRNAs (miRNAs) are endogenous small noncoding RNAs that regulate gene expression with functional links to tumorigenesis. Hepatocellular carcinoma (HCC) is the most common type of liver cancer, and it is heterogeneous in clinical outcomes and biological activities. Recently, we have identified a subset of highly invasive epithelial cell adhesion molecule (EpCAM)(+) HCC cells from alpha-fetoprotein (AFP)(+) tumors with cancer stem/progenitor cell features, that is, the abilities to self-renew, differentiate, and initiate aggressive tumors in vivo. Here, using a global microarray-based miRNA profiling approach followed by validation with quantitative reverse transcription polymerase chain reaction, we have demonstrated that conserved miR-181 family members were up-regulated in EpCAM(+)AFP(+) HCCs and in EpCAM(+) HCC cells isolated from AFP(+) tumors. Moreover, miR-181 family members were highly expressed in embryonic livers and in isolated hepatic stem cells. Importantly, inhibition of miR-181 led to a reduction in EpCAM(+) HCC cell quantity and tumor initiating ability, whereas exogenous miR-181 expression in HCC cells resulted in an enrichment of EpCAM(+) HCC cells. We have found that miR-181 could directly target hepatic transcriptional regulators of differentiation (for example, caudal type homeobox transcription factor 2 [CDX2] and GATA binding protein 6 [GATA6]) and an inhibitor of Wnt/beta-catenin signaling (nemo-like kinase [NLK]). Taken together, our results define a novel regulatory link between miR-181s and human EpCAM(+) liver cancer stem/progenitor cells and imply that molecular targeting of miR-181 may eradicate HCC.

Numerous studies of human gene function have been launched since the sequencing of the human genome. Global molecular profiling studies of hepatocellular carcinoma (HCC) are providing a comprehensive view of the expression changes that occur during the carcinogenic process and are uncovering promising biomarkers with clinical potential. In this chapter, an overview of recent gene expression profiling of human HCC is provided along with a summation of the mechanistic, diagnostic, and prognostic significance of these findings. Emerging concepts associated with these studies are also addressed and biomarkers present in serum are highlighted. Current profiling studies, conducted on multiple array platforms, are powerful tools which have provided useful clues to begin to unravel the mechanisms of HCC biology and improve clinical outcome.

To study the differential expression of the S100 gene family at the RNA level in human esophageal squamous cell carcinoma (ESCC), and to find the relationship of the S100 gene family with ESCC.Firstly, the specific primers were designed for the different S100 genes with Software Primer 3, which required that both primer sequences of each S100 gene were from two different exons respectively. Then, the differential expression of 16 S100 genes was examined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) in 62 cases of ESCC versus the corresponding normal esophageal mucosa. All RT-PCR products were analyzed by 1.5% agarose gel. With Fluor-S MultiImager and Multi-Analyst software, the electrophoresis images were evaluated with statistics analysis using SAS 8.1 software.Eleven out of 16 S100 genes were significantly downregulated ( p0.05) in ESCC versus the normal counterparts such as S100A1, S100A2, S100A4, S100A8, S100A9, S100A10, S100A11, S100A12, S100A14, S100B, and S100P genes. Only the S100A7 gene in the S100 family was markedly upregulated ( p0.05). Moreover, the S100B gene was significantly correlated with histological differentiation of ESCC ( p=0.0247), and the deregulation of some S100 genes was closely correlated ( p0.05), such as S100A10/S100A11, S100A2/S100A8, S100A2/S100A14, S100A8/S100A14, and S100A2/S100P etc.The S100 gene family is closely associated with ESCC.

Globally, hepatocellular carcinoma (HCC) accounts for 70-85% of primary liver cancers and ranks second in the 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. Using microarray-based global microRNA profiling in 223 HCC patients, we found that members of the miR-29 family were the most significantly (p Citation Format: Sonya T. Parpart, Stephanie Roessler, Fei Dong, Vinay Rao, Junfang Ji, Lun–Xiu Qin, Qing–Hai Ye, Zhao–You Tang, Hu–Liang Jia, Xin Wei Wang. Modulation of miR-29 expression by alpha-fetoprotein is linked to the hepatocellular carcinoma epigenome. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5183. doi:10.1158/1538-7445.AM2014-5183

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).

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.

Hepatocellular carcinoma (HCC) is a malignant cancer with the observable heterogeneity and microRNAs (miRNAs) are functional associated with the tumorignesis of HCC. We recently identified that a group of HCC cells isolated with epithelial cell adhesion molecular (EpCAM) antibody are hepatic cancer stem cells (CSCs). In EpCAM+ hepatic CSCs, all miR-181 family members are highly expressed and Wnt/beta-catenin signaling is activated. Here, we investigated whether Wnt/beta-catenin signaling could regulate miR-181s expression in HCC. We first detected the association of beta-catenin and miR-181s expression in vitro and in vivo, and found that they were positively correlated. Further, the expression of miR-181s was induced following the activation of Wnt/beta-catenin signaling by the employment of Wnt10B conditioned media, LiCl, and transfection of beta-catenin and Tcf4. Meanwhile, the level of miR-181s was decreased after the inactivation of Wnt/beta-catenin signaling with induction of adenomatosis polyposis coli and transfection of beta-catenin siRNA. Moreover, we found Tcf4 associated with the promoter region of miR-181a-2 and miR-181b-2, in which there existed 7 classic Tcf4 binding elements. Taken together, our data indicate that miR-181s could be activated by Wnt/beta-catenin signaling and the transcript of miR-181a-2 and miR-181b-2 is a novel target of Tcf4/beta-catenin complex in HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2030.