Dafang Wan

CD133 positive hepatocellular carcinoma cells possess high capacity for tumorigenicity.

Recently increasing reported data have suggested that only a small subset of cancer cells possess capability to initiate malignancies including leukemia and solid tumors, which was based on investigation in these cells displaying a distinct surface marker pattern within the primary cancers. CD133 is a putative hematopoietic and neuronal stem‐cell marker, which was also considered as a tumorigenic marker in brain and prostate cancer. We hypothesized that CD133 was a marker closely correlated with tumorigenicity, since it was reported that CD133 expressed in human fetal liver and repairing liver tissues, which tightly associated with hepatocarcinogenesis. Our findings showed that a small population of CD133 positive cells indeed exists in human hepatocellular carcinoma (HCC) cell lines and primary HCC tissues. From SMMC‐7721 cell line, CD133+ cells isolated by MACS manifested high tumorigenecity and clonogenicity as compared with CD133− HCC cells. The implication that CD133 might be one of the markers for HCC cancer stem‐like cells needed further investigation.

Diagnostic and prognostic implications of microRNAs in human hepatocellular carcinoma

MicroRNAs (miRNAs) are important gene regulators, which are often deregulated in cancers. In this study, the authors analyzed the microRNAs profiles of 78 matched cancer/noncanerous liver tissues from HCC patients and 10 normal liver tissues and found that 69 miRNAs were differentially expressed between hepatocellular carcinoma (HCC) and corresponding noncancerous liver tissues (N). Then the expressions of 8 differentially expressed miRNAs were validated by real time RT PCR. The set of differentially expressed miRNAs could distinctly classify HCC, N and normal liver tissues (NL). Moreover, some of these differentially expressed miRNAs were related to the clinical factors of HCC patients. Most importantly, Kaplan‐Meier estimates and the log‐rank test showed that high expression of hsa‐miR‐125b was correlated with good survival of HCC patients (hazard ratio, 1.787, 95% confidence interval, 1.020–3.133, p = 0.043). The transfection assay showed that overexpression of miR‐125b in HCC cell line could obviously suppress the cell growth and phosporylation of Akt. In conclusion, the authors have demonstrated the diagnostic miRNA profile for HCC, and for the first time, identified the miR‐125b with predictive significance for HCC prognosis.

Gain of miR-151 on chromosome 8q24.3 facilitates tumour cell migration and spreading through downregulating RhoGDIA

Recurrent chromosomal aberrations are often observed in hepatocellular carcinoma (HCC), but little is known about the functional non-coding sequences, particularly microRNAs (miRNAs), at the chromosomal breakpoints in HCC. Here we show that 22 miRNAs are often amplified or deleted in HCC. MicroRNA-151 (miR-151), a frequently amplified miRNA on 8q24.3, is correlated with intrahepatic metastasis of HCC. We further show that miR-151, which is often expressed together with its host gene FAK, encoding focal adhesion kinase, significantly increases HCC cell migration and invasion in vitro and in vivo, mainly through miR-151-5p, but not through miR-151-3p. Moreover, miR-151 exerts this function by directly targeting RhoGDIA, a putative metastasis suppressor in HCC, thus leading to the activation of Rac1, Cdc42 and Rho GTPases. In addition, miR-151 can function synergistically with FAK to enhance HCC cell motility and spreading. Thus, our findings indicate that chromosome gain of miR-151 is a crucial stimulus for tumour invasion and metastasis of HCC.

MicroRNA-30d promotes tumor invasion and metastasis by targeting Galphai2 in hepatocellular carcinoma†

The pathological relevance and significance of microRNAs (miRNAs) in hepatocarcinogenesis have attracted much attention in recent years; however, little is known about the underlying molecular mechanisms through which miRNAs are involved in the development and progression of hepatocellular carcinoma (HCC). In this study, we demonstrate that miR‐30d is frequently up‐regulated in HCC and that its expression is highly associated with the intrahepatic metastasis of HCC. Furthermore, the enhanced expression of miR‐30d could promote HCC cell migration and invasion in vitro and intrahepatic and distal pulmonary metastasis in vivo, while silencing its expression resulted in a reduced migration and invasion. Galphai2 (GNAI2) was identified as the direct and functional target of miR‐30d with integrated bioinformatics analysis and messenger RNA array assay. This regulation was further confirmed by luciferase reporter assays. In addition, our results, for the first time, showed that GNAI2 was frequently suppressed in HCC by way of quantitative reverse‐transcription polymerase chain reaction and immunohistochemical staining assays. The increase of the GNAI2 expression significantly inhibits, whereas knockdown of the GNAI2 expression remarkably enhances HCC cell migration and invasion, indicating that GNAI2 functions as a metastasis suppressor in HCC. The restoration of GNAI2 can inhibit miR‐30d–induced HCC cell invasion and metastasis. Conclusion: The newly identified miR‐30d/GNAI2 axis elucidates the molecular mechanism of HCC cell invasion and metastasis and represents a new potential therapeutic target for HCC treatment. (HEPATOLOGY 2010.)

Upregulation of miR-23a∼27a∼24 decreases transforming growth factor-beta-induced tumor-suppressive activities in human hepatocellular carcinoma cells

Transforming growth factor‐beta (TGF‐beta) plays a dual and complex role in human cancer. In this report, we observe a specific set of MicroRNAs (miRNAs) changed in response to TGF‐beta in human hepatocellular carcinoma (HCC) cells by miRNA microarray screening. A cluster of miRNA, miR‐23a∼27a∼24, is induced in an early stage by TGF‐beta in Huh‐7 cells. Knockdown of Smad4, Smad2 or Smad3 expression by RNA interference can attenuate the response of miR‐23a∼27a∼24 to TGF‐beta addition, indicating that this induction is dependent on Smad pathway. We also explore that miR‐23a∼27a∼24 can function as an antiapoptotic and proliferation‐promoting factor in liver cancer cells. In addition, expression of this miRNA cluster is found to be remarkably upregulated in HCC tissues versus normal liver tissues. These findings suggest a novel, alternative mechanism through which TGF‐beta could induce specific miRNA expression to escape from tumor‐suppressive response in HCC cells.

Upregulation of miR-23a approximately 27a approximately 24 decreases transforming growth factor-beta-induced tumor-suppressive activities in human hepatocellular carcinoma cells.

Transforming growth factor‐beta (TGF‐beta) plays a dual and complex role in human cancer. In this report, we observe a specific set of MicroRNAs (miRNAs) changed in response to TGF‐beta in human hepatocellular carcinoma (HCC) cells by miRNA microarray screening. A cluster of miRNA, miR‐23a∼27a∼24, is induced in an early stage by TGF‐beta in Huh‐7 cells. Knockdown of Smad4, Smad2 or Smad3 expression by RNA interference can attenuate the response of miR‐23a∼27a∼24 to TGF‐beta addition, indicating that this induction is dependent on Smad pathway. We also explore that miR‐23a∼27a∼24 can function as an antiapoptotic and proliferation‐promoting factor in liver cancer cells. In addition, expression of this miRNA cluster is found to be remarkably upregulated in HCC tissues versus normal liver tissues. These findings suggest a novel, alternative mechanism through which TGF‐beta could induce specific miRNA expression to escape from tumor‐suppressive response in HCC cells.

Acetylcholinesterase, a key prognostic predictor for hepatocellular carcinoma, suppresses cell growth and induces chemosensitization†

Acetylcholinesterase (ACHE) plays important roles in the cholinergic system, and its dysregulation is involved in a variety of human diseases. However, the roles and implications of ACHE in hepatocellular carcinoma (HCC) remain elusive. Here we demonstrate that ACHE was significantly down‐regulated in the cancerous tissues of 69.2% of HCC patients, and the low ACHE expression in HCC was correlated with tumor aggressiveness, an elevated risk of postoperative recurrence, and a low survival rate. Both the recombinant ACHE protein and the enhanced expression of ACHE significantly inhibited HCC cell growth in vitro and tumorigenicity in vivo. Further study showed that ACHE suppressed cell proliferation via its enzymatic activity of acetylcholine catalysis and degradation. Moreover, ACHE could inactivate mitogen‐activated protein kinase and phosphatidyl inositol‐3′‐phosphate kinase/protein kinase B pathways in HCC cells and thereby increase the activation of glycogen synthase kinase 3β and lead to β‐catenin degradation and cyclin D1 suppression. In addition, increased ACHE expression could remarkably sensitize HCC cells to chemotherapeutic drugs (i.e., adriamycin and etoposide). Conclusion: For the first time, we describe the function of ACHE as a tumor growth suppressor in regulating cell proliferation, the relevant signaling pathways, and the drug sensitivity of HCC cells. ACHE is a promising independent prognostic predictor for HCC recurrence and the survival of HCC patients. These findings provide new insights into potential strategies for drug discovery and improved HCC treatment. (HEPATOLOGY 2011;53:493‐503)

Analysis of acetylcholine, choline and butyrobetaine in human liver tissues by hydrophilic interaction liquid chromatography-tandem mass spectrometry.

The strong polar quaternary ammoniums, acetylcholine (ACh), choline (Ch) and butyrobetaine (BB, (3-carboxypropyl)trimethylammonium), are believed playing important roles in liver metabolism. These metabolites are at low levels and are weakly retained on reversed-phase liquid chromatographic (RP-LC) columns. Several hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) methods have been reported to analyze these compounds from different samples. However, no application to human liver tissues has been published. In this study, HILIC-MS/MS method was developed to simultaneously determine these three metabolites in human liver tissues. They were simply extracted from tissue, separated on a HILIC column, and detected by tandem MS in the mode of multiple reaction monitoring (MRM). Further studies on the recovery and repeatability based on real samples indicated the method was accurate and reliable. This method was successfully applied to measure the levels of ACh, Ch and BB in 61 human liver tissue samples including normal, hepatocellular carcinoma (HCC) and matched non-cancerous liver tissues. By comparison of Ch and ACh contents in 29 HCC with their matched non-cancerous liver tissues, it was found that ACh content increased in 11/29 HCC cases and decreased in 13/29 cases. Furthermore, the ACh/Ch ratio increased in 16/29 HCC cases, while it decreased in 8/29 cases. These results strongly indicated that there exist different patterns of ACh content in cancer tissues among HCC patients, thus highlighting the understanding of ACh and its relevant signal pathways in hepatic carcinogenesis and HCC progression.

The apoptosis-associated protein BNIPL interacts with two cell proliferation-related proteins, MIF and GFER.

Bcl‐2/adenovirus E1B 19 kDa interacting protein 2‐like, BNIP‐2‐like (BNIPL) is a recently cloned and characterized apoptosis‐associated protein that shares 72% homology with BNIP‐2. It is highly expressed in human placenta and lung. A yeast two‐hybrid system was used to obtain two BNIPL‐interacting proteins, MIF (macrophage migration inhibitory factor) and GFER (growth factor erv1 (Saccharomyces cerevisiae)‐like). The interactions were confirmed by glutathione S‐transferase pull‐down assay in vitro and co‐immunoprecipitation assay in vivo. Colony formation assay and cell proliferation test suggest that overexpression of BNIPL could inhibit the growth of BEL‐7402 cells. These findings suggest that BNIPL may physically bind to cell proliferation‐related proteins, MIF and GFER.

The minimum LOH region defined on chromosome 17p13.3 in human hepatocellular carcinoma with gene content analysis.

Hepatocellular carcinoma (HCC) is one of the most common human cancers in Asia. Previous studies have shown that in addition to aberrations of the p53 gene on chromosome 17p13.1, other gene(s) on chromosome 17p13.3 may also play a role in HCC. To detect the status of loss of heterozygosity (LOH) in HCC and to determine the minimum region of LOH on 17p13.3, we analyzed 22 paired HCC and non-cancerous liver samples with 14 polymorphic markers plus TP53 (p53 gene) as a comparison. The data revealed a high level of LOH (>68%) in a minimum region between D17S1866 and D17S1574, spanning over a 1.5 Mb region. Genomic library screening using markers in the region has resulted in the isolation of a cluster of BAC/PAC clones. We created a physical map in this region. Using large-scale genome sequencing, gene annotation, cDNA screening, and exon trapping, we identified 17 known genes and 13 novel genes in the minimum region. The function of these genes was analyzed and the possibility of several putative tumor suppressor genes was discussed.