Ling-Hao Zhao

Genomic and oncogenic preference of HBV integration in hepatocellular carcinoma

Hepatitis B virus (HBV) can integrate into the human genome, contributing to genomic instability and hepatocarcinogenesis. Here by conducting high-throughput viral integration detection and RNA sequencing, we identify 4,225 HBV integration events in tumour and adjacent non-tumour samples from 426 patients with HCC. We show that HBV is prone to integrate into rare fragile sites and functional genomic regions including CpG islands. We observe a distinct pattern in the preferential sites of HBV integration between tumour and non-tumour tissues. HBV insertional sites are significantly enriched in the proximity of telomeres in tumours. Recurrent HBV target genes are identified with few that overlap. The overall HBV integration frequency is much higher in tumour genomes of males than in females, with a significant enrichment of integration into chromosome 17. Furthermore, a cirrhosis-dependent HBV integration pattern is observed, affecting distinct targeted genes. Our data suggest that HBV integration has a high potential to drive oncogenic transformation.

Acetyl‐coenzyme A carboxylase alpha promotion of glucose‐mediated fatty acid synthesis enhances survival of hepatocellular carcinoma in mice and patients

Solid tumors often suffer from suboptimal oxygen and nutrient supplies. This stress underlies the requirement for metabolic adaptation. Aberrantly activated de novo lipogenesis is critical for development and progression of human hepatocellular carcinoma (HCC). However, whether de novo lipogenesis influences biological behaviors of HCCs under conditions of metabolic stress are still poorly understood. Here, we show that HCCs display distinct levels of glucose‐derived de novo lipogenesis, which are positively correlated with their survival responses to glucose limitation. The enhanced lipogenesis in HCCs is characterized by an increased expression of rate‐limiting enzyme acetyl‐coenzyme A carboxylase alpha (ACCα). ACCα‐mediated fatty acid (FA) synthesis determines the intracellular lipid content that is required to maintain energy hemostasis and inhibit cell death by means of FA oxidation (FAO) during metabolic stress. In accord, overexpression of ACCα facilitates tumor growth. ACCα forms a complex with carnitine palmitoyltransferase 1A (CPT1A) and prevents its mitochondria distribution under nutrient‐sufficient conditions. During metabolic stress, phosphorylation of ACCα leads to dissociation of the complex and mitochondria localization of CPT1A, thus promoting FAO‐mediated cell survival. Therefore, ACCα could provide both the substrate and enzyme storage for FAO during glucose deficiency. Up‐regulation of ACCα is also significantly correlated with poorer overall survival and disease recurrence postsurgery. Multivariate Coxs regression analysis identified ACCα as an effective predictor of poor prognosis. Conclusion: These results present novel mechanistic insight into a pivotal role of ACCα in maintaining HCC survival under metabolic stress. It could be exploited as a novel diagnostic marker and therapeutic target. (Hepatology 2016;63:1272–1286)

HBx regulates fatty acid oxidation to promote hepatocellular carcinoma survival during metabolic stress

Due to a high rate of nutrient consumption and inadequate vascularization, hepatocellular carcinoma (HCC) cells constantly undergo metabolic stress during tumor development. Hepatitis B virus (HBV) X protein (HBx) has been implicated in the pathogenesis of HBV-induced HCC. In this study, we investigated the functional roles of HBx in HCC adaptation to metabolic stress. Up-regulation of HBx increased the intracellular ATP and NADPH generation, and induced the resistance to glucose deprivation, whereas depletion of HBx via siRNA abolished these effects and conferred HCC cells sensitive to glucose restriction. Though HBx did not affect the glycolysis and oxidative phosphorylation capacity of HCC cells under normal culture conditions, it facilitated fatty acid oxidation (FAO) in the absence of glucose, which maintained NADPH and ATP levels. Further investigation showed that HBx expression, under glucose deprivation, stimulated phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) via a calcium/CaMKK-dependent pathway, which was required for the activation of FAO. Conversely, inhibition of FAO by etomoxir (ETO) restored the sensitivity of HBx-expressing cells to glucose deficiency in vitro and retarded xenograft tumor formation in vivo. Finally, HBx-induced activation of the AMPK and FAO pathways were also observed in xenograft tumors and HBV-associated HCC specimens. Our data suggest that HBx plays a key role in the maintenance of redox and energy homeostasis by activating FAO, which is critical for HCC cell survival under conditions of metabolic stress and might be exploited for therapeutic benefit.

Ligand-Directed Stearic Acid Grafted Chitosan Micelles to Increase Therapeutic Efficacy in Hepatic Cancer

Targeted delivery system would be an interesting platform to enhance the therapeutic effect and to reduce the side effects of anticancer drugs. In this study, we have developed lactobionic acid (LA)-modified chitosan-stearic acid (CS-SA) (CSS-LA) to deliver doxorubicin (DOX) to hepatic cancer cells. The average particle size of CSS-LA/DOX was ∼100 nm with a high entrapment efficiency of >95%. Drug release studies showed that DOX release from pH-sensitive micelles is significantly faster at pH 5.0 than at pH 7.4. The LA conjugated micelles showed enhanced cellular uptake in HepG2 and BEL-7402 liver cancer cells than free drug and unconjugated micelles. Consistently, CSS-LA/DOX showed enhanced cell cytotoxicity in these two cell lines. Annexin-V/FITC and PI based apoptosis assay showed that the number of living cells greatly reduced in this group with marked presence of necrotic and apoptotic cells. LA-conjugated carrier induced typical chromatic condensation of cells; membrane blebbing and apoptotic bodies began to appear. In vivo, CSS-LA/DOX showed an excellent tumor regression profile with no toxic side effects. The active targeting moiety, long circulation profile, and EPR effect contributed to its superior anticancer effect in HepG2 based tumor. Our results showed that polymeric micelles conjugated with LA increased the therapeutic availability of DOX in the liver cancer cell based solid tumor without any toxic side effects. The active targeting ligand conjugated nanoparticulate system could be a promising therapeutic strategy in the treatment of hepatic cancers.

Pioglitazone, a PPARγ agonist, inhibits growth and invasion of human hepatocellular carcinoma via blockade of the rage signaling.

Pioglitazone (PGZ), a synthetic PPARγ ligand, is known to have anti‐tumor activity. However, it is unclear how it acts against hepatocellular carcinoma (HCC). We hypothesized that the pathological receptor for advanced glycation end products (RAGE) is involved in the PGZ anti‐tumor process. To test this notion, human primary HCC tissues and corresponding adjacent non‐cancerous tissues (ANCT) from 75 consecutive cases were analyzed. The expression and clinical significance of RAGE was assessed by immunohistochemical assay through tissue microarray. After HCC cells were pretreated with different concentrations of PGZ, cell proliferation, apoptosis, cell invasion, and cell cycle distribution were evaluated by multiple assays. The results showed that, the positive expression of RAGE was significantly higher in HCC tissues than in ANCT (66.7% vs. 36.0%, P < 0.001), and was closely associated with pathological staging (P = 0.014) and lymph‐vascular space invasion (P = 0.003). Moreover, PGZ inhibited proliferative activity and invasive potential, and induced apoptosis and cell cycle arrest in HCC cells resulting in increased expression of PPARγ and decreased expression of RAGE, NF‐κB, HMGB1, p38MAPK, Ki‐67, MMP‐2, and CyclinD1. Furthermore, knockdown of RAGE or NF‐κB by siRNA effectively suppressed cell proliferation and invasion, and mediated the inhibitory effects of PGZ in HCC cells. Taken together, our findings suggest that, RAGE is overexpressed in human HCC tissues, and is closely associated with the pathological staging and tumor invasion of HCC. In addition, PGZ as a PPARγ agonist may inhibit growth and invasion of HCC cells via blockade of the RAGE signaling.

Aldehyde dehydrogenase‐2 (ALDH2) opposes hepatocellular carcinoma progression by regulating AMP‐activated protein kinase signaling in mice

Potential biomarkers that can be used to determine prognosis and perform targeted therapies are urgently needed to treat patients with hepatocellular carcinoma (HCC). To meet this need, we performed a screen to identify functional genes associated with hepatocellular carcinogenesis and its progression at the transcriptome and proteome levels. We identified aldehyde dedydrogenase‐2 (ALDH2) as a gene of interest for further study. ALDH2 levels were significantly lower at the mRNA and protein level in tumor tissues than in normal tissues, and they were even lower in tissues that exhibited increased migratory capacity. A study of clinical associations showed that ALDH2 is correlated with survival and multiple migration‐associated clinicopathological traits, including the presence of metastasis and portal vein tumor thrombus. The result of overexpressing or knocking down ALDH2 showed that this gene inhibited migration and invasion both in vivo and in vitro. We also found that ALDH2 altered the redox status of cells by regulating acetaldehyde levels and that it further activated the AMP‐activated protein kinase (AMPK) signaling pathway. Conclusion: Decreased levels of ALDH2 may indicate a poor prognosis in HCC patients, while forcing the expression of ALDH2 in HCC cells inhibited their aggressive behavior in vitro and in mice largely by modulating the activity of the ALDH2‐acetaldehyde‐redox‐AMPK axis. Therefore, identifying ALDH2 expression levels in HCC might be a useful strategy for classifying HCC patients and for developing potential therapeutic strategies that specifically target metastatic HCC. (Hepatology 2017;65:1628‐1644).

Neuraminidase 1 (NEU1) promotes proliferation and migration as a diagnostic and prognostic biomarker of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is among the most malignant cancers worldwide, lacking biomarkers for subtyping and the reliable prognostication. Herein, we report a novel biomarker, NEU1 (neuraminidase 1), is up-regulated in most samples of HCC. The diagnostic value of NEU1 was evaluated by ROC, and the AUC (area under curve) reached 0.87 and 0.96 in two independent datasets, respectively. The survival differences of HCC patients with high or low expression of NEU1 were statistically significant, and a significant correlation between NEU1 expression and clinical information including stage, differentiation, AFP and embolus were observed. NEU1 expression, at both the mRNA and protein levels, were also higher in the portal vein tumor thrombus than tumor tissues. We also measured the proliferation and migration ability of two HCC cell lines following NEU1 interference and over-expression. Migration and proliferation rate were increased in NEU1 high expression groups. Moreover, gene expression studies identified pathways significantly associated with NEU1 expression. Among them, all the genes involved in spliceosomepathway were up regulated in NEU1-high group. In summary, our work identified NEU1 as a novel biomarker for both diagnosis and prognosis in HCC, and one of the most altered pathway of NEU1 is spliceosome.

A New Approach to Evaluating Aberrant DNA Methylation Profiles in Hepatocellular Carcinoma as Potential Biomarkers

Hypermethylation of CpG islands in the promoter region of tumor suppressor genes (TSGs) and their subsequent silencing is thought to be one of the main mechanisms of carcinogenesis. MBD2b enrichment coupled with a NimbleGen array was applied to examine the genome-wide CpG island methylation profile of hepatocellular carcinoma (HCC). Hypermethylated DNA of 58 pairs of HCC and adjacent tissue samples was enriched and hybridized in the same array. Aberrant hypermethylated peaks of HCC and adjacent tissues were screened and annotated after data processing using NimbleScan2.5 and our newly developed Weighting and Scoring (WAS) method, respectively. Validation using bisulfite sequencing of randomly selected ANKRD45, APC, CDX1, HOXD3, PTGER and TUBB6 genes demonstrated significant hypermethylation modification in HCC samples, consistent with the array data.