Xin Yuan Guan

Mutations of a mutS homolog in hereditary nonpolyposis colorectal cancer

Recent studies have shown that a locus responsible for hereditary nonpolyposis colorectal cancer (HNPCC) is on chromosome 2p and that tumors developing in these patients contain alterations in microsatellite sequences (RER+ phenotype). We have used chromosome microdissection to obtain highly polymorphic markers from chromosome 2p16. These and other markers were ordered in a panel of somatic cell hybrids and used to define a 0.8 Mb interval containing the HNPCC locus. Candidate genes were then mapped, and one was found to lie within the 0.8 Mb interval. We identified this candidate by virtue of its homology to mutS mismatch repair genes. cDNA clones were obtained and the sequence used to detect germline mutations, including those producing termination codons, in HNPCC kindreds. Somatic as well as germline mutations of the gene were identified in RER+ tumor cells. This mutS homolog is therefore likely to be responsible for HNPCC.

CD133+ HCC cancer stem cells confer chemoresistance by preferential expression of the Akt/PKB survival pathway

The recent discovery of cancer stem cells (CSCs) has played a pivotal role in changing our view of carcinogenesis and chemotherapy. Based on this concept, CSCs are responsible for the formation and growth of neoplastic tissue and are naturally resistant to chemotherapy, explaining why traditional chemotherapies can initially shrink a tumor but fails to eradicate it in full, allowing eventual recurrence. Recently, we identified a CSC population in hepatocellular carcinoma (HCC) characterized by their CD133 phenotype. However, the molecular mechanism by which it escapes conventional therapies remains unknown. Here, we examined the sensitivity of these cells to chemotherapeutic agents (doxorubicin and fluorouracil) and the possible mechanistic pathway by which resistance may be regulated. Purified CD133+ HCC cells isolated from human HCC cell line and xenograft mouse models survived chemotherapy in increased proportions relative to most tumor cells which lack the CD133 phenotype; the underlying mechanism of which required the preferential expression of survival proteins involved in the Akt/PKB and Bcl-2 pathway. Treatment of CD133+ HCC cells with an AKT1 inhibitor, specific to the Akt/PKB pathway, significantly reduced the expression of the survival proteins that was normally expressed endogenously. In addition, treatment of unsorted HCC cells with both anticancer drugs in vitro significantly enriched the CD133+ subpopulation. In conclusion, our results show that CD133+ HCC cells contribute to chemoresistance through preferential activation of Akt/PKB and Bcl-2 cell survival response. Targeting of this specific survival signaling pathway in CD133+ HCC CSCs may provide a novel therapeutic model for the disease.

Genomic instability in laminopathy-based premature aging

Premature aging syndromes often result from mutations in nuclear proteins involved in the maintenance of genomic integrity. Lamin A is a major component of the nuclear lamina and nuclear skeleton. Truncation in lamin A causes Hutchinson-Gilford progerial syndrome (HGPS), a severe form of early-onset premature aging. Lack of functional Zmpste24, a metalloproteinase responsible for the maturation of prelamin A, also results in progeroid phenotypes in mice and humans. We found that Zmpste24-deficient mouse embryonic fibroblasts (MEFs) show increased DNA damage and chromosome aberrations and are more sensitive to DNA-damaging agents. Bone marrow cells isolated from Zmpste24−/− mice show increased aneuploidy and the mice are more sensitive to DNA-damaging agents. Recruitment of p53 binding protein 1 (53BP1) and Rad51 to sites of DNA lesion is impaired in Zmpste24−/− MEFs and in HGPS fibroblasts, resulting in delayed checkpoint response and defective DNA repair. Wild-type MEFs ectopically expressing unprocessible prelamin A show similar defects in checkpoint response and DNA repair. Our results indicate that unprocessed prelamin A and truncated lamin A act dominant negatively to perturb DNA damage response and repair, resulting in genomic instability which might contribute to laminopathy-based premature aging.

Aldehyde Dehydrogenase Discriminates the CD133 Liver Cancer Stem Cell Populations

Recent efforts in our study of cancer stem cells (CSC) in hepatocellular carcinoma (HCC) have led to the identification of CD133 as a prominent HCC CSC marker. Findings were based on experiments done on cell lines and xenograft tumors where expression of CD133 was detected at levels as high as 65%. Based on the CSC theory, CSCs are believed to represent only a minority number of the tumor mass. This is indicative that our previously characterized CD133+ HCC CSC population is still heterogeneous, consisting of perhaps subsets of cells with differing tumorigenic potential. We hypothesized that it is possible to further enrich the CSC population by means of additional differentially expressed markers. Using a two-dimensional PAGE approach, we compared protein profiles between CD133+ and CD133− subpopulations isolated from Huh7 and PLC8024 and identified aldehyde dehydrogenase 1A1 as one of the proteins that are preferentially expressed in the CD133+ subfraction. Analysis of the expression of several different ALDH isoforms and ALDH enzymatic activity in liver cell lines found ALDH to be positively correlated with CD133 expression. Dual-color flow cytometry analysis found the majority of ALDH+ to be CD133+, yet not all CD133+ HCC cells were ALDH+. Subsequent studies on purified subpopulations found CD133+ALDH+ cells to be significantly more tumorigenic than their CD133−ALDH+ or CD133−ALDH− counterparts, both in vitro and in vivo. These data, combined with those from our previous work, reveal the existence of a hierarchical organization in HCC bearing tumorigenic potential in the order of CD133+ALDH+ > CD133+ALDH− > CD133−ALDH−. ALDH, expressed along CD133, can more specifically characterize the tumorigenic liver CSC population. (Mol Cancer Res 2008;6(7):1146–53)

Twist Overexpression Correlates with Hepatocellular Carcinoma Metastasis through Induction of Epithelial-Mesenchymal Transition

Purpose: Hepatocellular carcinoma (HCC) is a rapidly growing tumor associated with a high propensity for vascular invasion and metastasis. Epithelial-mesenchymal transition (EMT) is a key event in the tumor invasion process. Recently, Twist has been identified to play an important role in EMT-mediated metastasis through the regulation of E-cadherin expression. However, the actual role of Twist in tumor invasiveness remains unclear. The purpose of this study is to investigate the expression and possible role of Twist in HCC. Experimental Design: We evaluated Twist and E-cadherin expression in HCC tissue microarray of paired primary and metastatic HCC by immunohistochemical staining. The role of Twist in EMT-mediated invasiveness was also evaluated in vitro in HCC cell lines. Results: We first showed that overexpression of Twist was correlated with HCC metastasis (P = 0.001) and its expression was negatively correlated with E-cadherin expression (P = 0.001, r = −0.443) by tissue microarray. A significant increase of Twist at the mRNA level was also found in metastatic HCC cell lines MHCC-97H, MHCC-97L, and H2M when compared with nonmetastatic Huh-7, H2P, and PLC cell lines. The MHCC-97H cell line, which has a higher metastatic ability, was found to have a higher level of Twist than MHCC-97L. Accompanied with increased Twist expression in the metastatic HCC cell lines when compared with the nonmetastatic primary ones, we found decreased E-cadherin mRNA expression in the metastatic HCC cell lines. By ectopic transfection of Twist into PLC cells, Twist was able to suppress E-cadherin expression and induce EMT changes, which was correlated with increased HCC cell invasiveness. Conclusion: This study shows that Twist overexpression was correlated with HCC metastasis through induction of EMT changes and HCC cell invasiveness.

miR-130b Promotes CD133+ Liver Tumor-Initiating Cell Growth and Self-Renewal via Tumor Protein 53-Induced Nuclear Protein 1

A novel paradigm in tumor biology suggests that cancer growth is driven by stem-like cells within a tumor, called tumor-initiating cells (TICs) or cancer stem cells (CSCs). Here we describe the identification and characterization of such cells from hepatocellular carcinoma (HCC) using the marker CD133. CD133 accounts for approximately 1.3%-13.6% of the cells in the bulk tumor of human primary HCC samples. When compared with their CD133⁻ counterparts, CD133(+) cells not only possess the preferential ability to form undifferentiated tumor spheroids in vitro but also express an enhanced level of stem cell-associated genes, have a greater ability to form tumors when implanted orthotopically in immunodeficient mice, and can be serially passaged into secondary animal recipients. Xenografts resemble the original human tumor and maintain a similar percentage of tumorigenic CD133(+) cells. Quantitative PCR analysis of 41 separate HCC tissue specimens with follow-up data found that CD133(+) tumor cells were frequently detected at low quantities in HCC, and their presence was also associated with worse overall survival and higher recurrence rates. Subsequent differential microRNA expression profiling of CD133(+) and CD133⁻ cells from human HCC clinical specimens and cell lines identified an overexpression of miR-130b in CD133(+) TICs. Functional studies on miR-130b lentiviral-transduced CD133⁻ cells demonstrated superior resistance to chemotherapeutic agents, enhanced tumorigenicity in vivo, and a greater potential for self renewal. Conversely, antagonizing miR-130b in CD133(+) TICs yielded an opposing effect. The increased miR-130b paralleled the reduced TP53INP1, a known miR-130b target. Silencing TP53INP1 in CD133⁻ cells enhanced both self renewal and tumorigenicity in vivo. Collectively, miR-130b regulates CD133(+) liver TICs, in part, via silencing TP53INP1.

The putative tumour suppressor microRNA-124 modulates hepatocellular carcinoma cell aggressiveness by repressing ROCK2 and EZH2

Background Recent profile studies of microRNA (miRNA) expression have documented a deregulation of miRNA (miR-124) in hepatocellular carcinoma (HCC). Objective To determine the status of miR-124 expression and its underlying mechanisms in the pathogenesis of HCC. Methods The expression levels of miR-124 were first examined in HCC cell lines and tumour tissues by real-time PCR. The in vitro and in vivo functional effect of miR-124 was examined further. A luciferase reporter assay was conducted to confirm target associations. Results The expression levels of miR-124 were frequently reduced in HCC cells and tissues, and low-level expression of miR-124 was significantly associated with a more aggressive and/or poor prognostic phenotype of patients with HCC (p<0.05). In HCC cell lines, stable overexpression of miR-124 was sufficient to inhibit cell motility and invasion in vitro, and suppress intrahepatic and pulmonary metastasis in vivo. In addition, ectopic overexpression of miR-124 in HCC cells inhibited epithelial–mesenchymal cell transition, formation of stress fibres, filopodia and lamellipodia. Further studies showed that miR-124 could directly target the 3′-untranslated region (3′-UTR) of both ROCK2 and EZH2 mRNAs, and suppress their mRNA and protein expressions. These findings suggest that miR-124 plays a critical role in regulating cytoskeletal events and epithelial–mesenchymal cell transition and, ultimately, inhibits the invasive and/or metastatic potential of HCC, probably by its direct target on ROCK2 and EZH2 genes. These results provide functional and mechanistic links between the tumour suppressor miRNA-124 and the two oncogenes ROCK2 and EZH2 on the aggressive nature of HCC. Conclusion These data highlight an important role for miR-124 in the regulation of invasion and metastasis in the molecular aetiology of HCC, and suggest a potential application of miR-124 in prognosis prediction and cancer treatment.

A Nuclear Factor, ASC-2, as a Cancer-amplified Transcriptional Coactivator Essential for Ligand-dependent Transactivation by Nuclear Receptors in Vivo

Many transcription coactivators interact with nuclear receptors in a ligand- and C-terminal transactivation function (AF2)-dependent manner. We isolated a nuclear factor (designated ASC-2) with such properties by using the ligand-binding domain of retinoid X receptor as a bait in a yeast two-hybrid screening. ASC-2 also interacted with other nuclear receptors, including retinoic acid receptor, thyroid hormone receptor, estrogen receptor α, and glucocorticoid receptor, basal factors TFIIA and TBP, and transcription integrators CBP/p300 and SRC-1. In transient cotransfections, ASC-2, either alone or in conjunction with CBP/p300 and SRC-1, stimulated ligand-dependent transactivation by wild type nuclear receptors but not mutant receptors lacking the AF2 domain. Consistent with an idea that ASC-2 is essential for the nuclear receptor function in vivo, microinjection of anti-ASC-2 antibody abrogated the ligand-dependent transactivation of retinoic acid receptor, and this repression was fully relieved by coinjection of ASC-2-expression vector. Surprisingly, ASC-2 was identical to a gene previously identified during a search for genes amplified and overexpressed in breast and other human cancers. From these results, we concluded that ASC-2 is a bona fidetranscription coactivator molecule of nuclear receptors, and its altered expression may contribute to the development of cancers.

Recoding RNA editing of AZIN1 predisposes to hepatocellular carcinoma

A better understanding of human hepatocellular carcinoma (HCC) pathogenesis at the molecular level will facilitate the discovery of tumor-initiating events. Transcriptome sequencing revealed that adenosine-to-inosine (A→I) RNA editing of AZIN1 (encoding antizyme inhibitor 1) is increased in HCC specimens. A→I editing of AZIN1 transcripts, specifically regulated by ADAR1 (encoding adenosine deaminase acting on RNA-1), results in a serine-to-glycine substitution at residue 367 of AZIN1, located in β-strand 15 (β15) and predicted to cause a conformational change, induced a cytoplasmic-to-nuclear translocation and conferred gain-of-function phenotypes that were manifested by augmented tumor-initiating potential and more aggressive behavior. Compared with wild-type AZIN1 protein, the edited form has a stronger affinity to antizyme, and the resultant higher AZIN1 protein stability promotes cell proliferation through the neutralization of antizyme-mediated degradation of ornithine decarboxylase (ODC) and cyclin D1 (CCND1). Collectively, A→I RNA editing of AZIN1 may be a potential driver in the pathogenesis of human cancers, particularly HCC.

MicroRNA‐29b suppresses tumor angiogenesis, invasion, and metastasis by regulating matrix metalloproteinase 2 expression

Hepatocellular carcinoma (HCC) is a highly vascularized tumor with frequent intrahepatic metastasis. Active angiogenesis and metastasis are responsible for rapid recurrence and poor survival of HCC. We previously found that microRNA‐29b (miR‐29b) down‐regulation was significantly associated with poor recurrence‐free survival of HCC patients. Therefore, the role of miR‐29b in tumor angiogenesis, invasion, and metastasis was further investigated in this study using in vitro capillary tube formation and transwell assays, in vivo subcutaneous and orthotopic xenograft mouse models, and Matrigel plug assay, and human HCC samples. Both gain‐ and loss‐of‐function studies showed that miR‐29b dramatically suppressed the ability of HCC cells to promote capillary tube formation of endothelial cells and to invade extracellular matrix gel in vitro. Using mouse models, we revealed that tumors derived from miR‐29b‐expressed HCC cells displayed significant reduction in microvessel density and in intrahepatic metastatic capacity compared with those from the control group. Subsequent investigations revealed that matrix metalloproteinase‐2 (MMP‐2) was a direct target of miR‐29b. The blocking of MMP‐2 by neutralizing antibody or RNA interference phenocopied the antiangiogenesis and antiinvasion effects of miR‐29b, whereas introduction of MMP‐2 antagonized the function of miR‐29b. We further disclosed that miR‐29b exerted its antiangiogenesis function, at least partly, by suppressing MMP‐2 expression in tumor cells and, in turn, impairing vascular endothelial growth factor receptor 2‐signaling in endothelial cells. Consistently, in human HCC tissues and mouse xenograft tumors miR‐29b level was inversely correlated with MMP‐2 expression, as well as tumor angiogenesis, venous invasion, and metastasis. Conclusion: miR‐29b deregulation contributes to angiogenesis, invasion, and metastasis of HCC. Restoration of miR‐29b represents a promising new strategy in anti‐HCC therapy. (HEPATOLOGY 2011;)