The diagnostic significance of CDH17-positive circulating tumor cells in patients with colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is the second leading cause of cancer deaths in Hong Kong. We tested the hypothesis that circulating tumor cell (CTC) analysis by ARB101 antibody could be used as a tool for CRC detection, progression, and therapy response. RESEARCH METHODS: ARB101 antibody was used for investigation of CDH17 expression in formalin-fixed, paraffin-embedded (FFPE) tissue sections and circulating tumor cells (CTCs) of CRC patients. RESULTS: Using ARB101, highest sensitivity was observed in 98/100 (98%) colorectal cancer tissue compared to 72/100 gastric cancer (72%) and 27/32 pancreatic cancer (84%). Immunoreactivity of CDH17 was significantly higher in distant metastatic (tumor-node-metastasis [TNM] stage IV) than non-distant metastatic (TNM stage I to III) CRC. ARB101 antibody also manifested the higher sensitivity than c-erbB2 (8%) and epidermal growth factor receptor (EGFR)-targeting antibodies (37%) with the significance (p < 0.0001). ARB101 positive CTCs were detected in 64/83 (77%) TNM stage I to IV CRC patients. Furthermore, ARB101 positive CTCs detected in TNM stage I to III CRC patients before and after surgical operation are statistically significant (p < 0.0001). CONCLUSIONS: CTC detection by ARB101 antibody could serve as a potential non-invasive approach for CRC detection, progression, and monitoring of treatment response.

Clinical correlation of cadherin-17 marker with advanced tumor stages and poor prognosis of cholangiocarcinoma.

BACKGROUND AND OBJECTIVES: In this retrospective study, we examined the CA17 tissue expression and analyzed its clinical significance in cholangiocarcinoma (CCA). MATERIALS AND METHODS: Immunohistochemistry was performed to assess CA17 expression on tissue microarrays in a training cohort enrolling 120 CCA patients and a validation cohort comprising 60 CCA patients. Image pro plus was applied to score the staining intensity and expression level of CA17 marker. Kaplan-Meier analysis, Cox's proportional hazards regression, and nomogram were applied to evaluate the prognostic significance of CA17. RESULTS: CA17 cancer biomarker over-expression was significantly observed in CCA compared to their non-tumor counterparts, and positively correlated with aggressive tumor phenotypes, like lymph node metastasis. Meanwhile, patients with high expression of CA17 correlated with worse postoperative overall survival (OS) and recurrence-free survival. Besides, multivariate analysis identified that CA17 expression was an independent prognostic factor for cholangiocarcinoma patients, which indicated that the CA17 could be more efficient than serum CA19-9 in predicting the OS of CCA patients. Notably, the nomogram integrating CA17 expression had better prognostic performance as compared with current TNM staging systems. CONCLUSION: CA17 was an independent adverse prognostic factor for CCA patients' survival, which may serve as a promising prognostic biomarker for CCA patients.

Integrin α2ß1 inhibits MST1 kinase phosphorylation and activates Yes-associated protein oncogenic signaling in hepatocellular carcinoma.

The Hippo pathway regulates the down-stream target Yes-associated protein (YAP) to maintain organ homeostasis, which is commonly inactivated in many types of cancers. However, how cell adhesion dysregulates the Hippo pathway activating YAP oncogene in hepatocellular carcinoma (HCC) remains unclear. Our findings demonstrate that α2ß1 integrin (but not other ß1 integrins) expressed in HCC cells, after binding to collagen extracellular matrix, could inhibit MST1 kinase phosphorylation and activate YAP pro-oncogenic activities. Knockdown of integrin α2 gene (ITGA2) suppressed YAP targeted gene expression in vitro. α2ß1 and collagen binding resulted in suppressing Hippo signaling of mammalian sterile 20-like kinase 1 (MST1) and Large tumor suppressor homolog 1 (LATS1) with concomitant activation of YAP-mediated connective tissue growth factor (CTGF) gene expression. In vitro kinase assay showed that MST1 is an immediate downstream target of integrin α2 with S1180 residue as the critical phosphorylation site. Clinical correlational analysis using a gene expression dataset of 228 HCC tumors revealed that ITGA2 expression was significantly associated with tumor progression, and co-expression with YAP targeted genes (AXL receptor tyrosine kinase, CTGF, cyclin D1, glypican 3, insulin like growth factor 1 receptor, and SRY-box 4) correlated with survivals of HCC patients. In conclusion, α2ß1 integrin activation through cellular adhesion impacts the Hippo pathway in solid tumors and modulates MST1-YAP signaling cascade. Targeting integrin α2 holds promises for treating YAP-positive HCC.

Diverse modes of genomic alteration in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a heterogeneous disease with high mortality rate. Recent genomic studies have identified TP53, AXIN1, and CTNNB1 as the most frequently mutated genes. Lower frequency mutations have been reported in ARID1A, ARID2 and JAK1. In addition, hepatitis B virus (HBV) integrations into the human genome have been associated with HCC. RESULTS: Here, we deep-sequence 42 HCC patients with a combination of whole genome, exome and transcriptome sequencing to identify the mutational landscape of HCC using a reasonably large discovery cohort. We find frequent mutations in TP53, CTNNB1 and AXIN1, and rare but likely functional mutations in BAP1 and IDH1. Besides frequent hepatitis B virus integrations at TERT, we identify translocations at the boundaries of TERT. A novel deletion is identified in CTNNB1 in a region that is heavily mutated in multiple cancers. We also find multiple high-allelic frequency mutations in the extracellular matrix protein LAMA2. Lower expression levels of LAMA2 correlate with a proliferative signature, and predict poor survival and higher chance of cancer recurrence in HCC patients, suggesting an important role of the extracellular matrix and cell adhesion in tumor progression of a subgroup of HCC patients. CONCLUSIONS: The heterogeneous disease of HCC features diverse modes of genomic alteration. In addition to common point mutations, structural variations and methylation changes, there are several virus-associated changes, including gene disruption or activation, formation of chimeric viral-human transcripts, and DNA copy number changes. Such a multitude of genomic events likely contributes to the heterogeneous nature of HCC.

Inhibition of STAT3 dimerization and acetylation by garcinol suppresses the growth of human hepatocellular carcinoma in vitro and in vivo.

BACKGROUND: Constitutive activation of signal transducer and activator of transcription 3 (STAT3) has been linked with proliferation, survival, invasion and angiogenesis of a variety of human cancer cells, including hepatocellular carcinoma (HCC). Thus, novel agents that can suppress STAT3 activation have potential for both prevention and treatment of HCC. Here we report, garcinol, a polyisoprenylated benzophenone, could suppress STAT3 activation in HCC cell lines and in xenografted tumor of HCC in nude mice model. EXPERIMENTAL DESIGN: Different HCC cell lines have been treated with garcinol and the inhibition of STAT3 activation, dimerization and acetylation have been checked by immunoblotting, immuno-fluorescence, and DNA binding assays. Xenografted tumor model has been generated in nude mice using HCC cell line and effect of garcinol in the inhibition of tumor growth has been investigated. RESULTS: Garcinol could inhibit both constitutive and interleukin (IL-6) inducible STAT3 activation in HCC cells. Computational modeling showed that garcinol could bind to the SH2 domain of STAT3 and suppress its dimerization in vitro. Being an acetyltransferase inhibitor, garcinol also inhibits STAT3 acetylation and thus impairs its DNA binding ability. The inhibition of STAT3 activation by garcinol led to the suppression of expression of various genes involved in proliferation, survival, and angiogenesis. It also suppressed proliferation and induced substantial apoptosis in HCC cells. Remarkably, garcinol inhibited the growth of human HCC xenograft tumors in athymic nu/nu mice, through the inhibition of STAT3 activation. CONCLUSION: Overall, our results suggest that garcinol exerts its anti-proliferative and pro-apoptotic effects through suppression of STAT3 signaling in HCC both in vitro and in vivo.

miR-122 targets pyruvate kinase M2 and affects metabolism of hepatocellular carcinoma.

In contrast to normal differentiated cells that depend on mitochondrial oxidative phosphorylation for energy production, cancer cells have evolved to utilize aerobic glycolysis (Warburg's effect), with benefit of providing intermediates for biomass production. MicroRNA-122 (miR-122) is highly expressed in normal liver tissue regulating a wide variety of biological processes including cellular metabolism, but is reduced in hepatocellular carcinoma (HCC). Overexpression of miR-122 was shown to inhibit cancer cell proliferation, metastasis, and increase chemosensitivity, but its functions in cancer metabolism remains unknown. The present study aims to identify the miR-122 targeted genes and to investigate the associated regulatory mechanisms in HCC metabolism. We found the ectopic overexpression of miR-122 affected metabolic activities of HCC cells, evidenced by the reduced lactate production and increased oxygen consumption. Integrated gene expression analysis in a cohort of 94 HCC tissues revealed miR-122 level tightly associated with a battery of glycolytic genes, in which pyruvate kinase (PK) gene showed the strongest anti-correlation coefficient (Pearson r = -0.6938, p = <0.0001). In addition, reduced PK level was significantly associated with poor clinical outcomes of HCC patients. We found isoform M2 (PKM2) is the dominant form highly expressed in HCC and is a direct target of miR-122, as overexpression of miR-122 reduced both the mRNA and protein levels of PKM2, whereas PKM2 re-expression abrogated the miR-122-mediated glycolytic activities. The present study demonstrated the regulatory role of miR-122 on PKM2 in HCC, having an implication of therapeutic intervention targeting cancer metabolic pathways.

Oncofetal gene SALL4 in aggressive hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma is the third leading cause of cancer-related deaths worldwide. In the heterogeneous group of hepatocellular carcinomas, those with characteristics of embryonic stem-cell and progenitor-cell gene expression are associated with the worst prognosis. The oncofetal gene SALL4, a marker of a subtype of hepatocellular carcinoma with progenitor-like features, is associated with a poor prognosis and is a potential target for treatment. METHODS: We screened specimens obtained from patients with primary hepatocellular carcinoma for the expression of SALL4 and carried out a clinicopathological analysis. Loss-of-function studies were then performed to evaluate the role of SALL4 in hepatocarcinogenesis and its potential as a molecular target for therapy. To assess the therapeutic effects of a peptide that targets SALL4, we used in vitro functional and in vivo xenograft assays. RESULTS: SALL4 is an oncofetal protein that is expressed in the human fetal liver and silenced in the adult liver, but it is reexpressed in a subgroup of patients who have hepatocellular carcinoma and an unfavorable prognosis. Gene-expression analysis showed the enrichment of progenitor-like gene signatures with overexpression of proliferative and metastatic genes in SALL4-positive hepatocellular carcinomas. Loss-of-function studies confirmed the critical role of SALL4 in cell survival and tumorigenicity. Blocking SALL4-corepressor interactions released suppression of PTEN (the phosphatase and tensin homologue protein) and inhibited tumor formation in xenograft models in vivo. CONCLUSIONS: SALL4 is a marker for a progenitor subclass of hepatocellular carcinoma with an aggressive phenotype. The absence of SALL4 expression in the healthy adult liver enhances the potential of SALL4 as a treatment target in hepatocellular carcinoma. (Funded by the Singapore National Medical Research Council and others.).

Circulating markers for prognosis of hepatocellular carcinoma.

INTRODUCTION: Hepatocellular carcinoma (HCC) is a lethal liver malignancy with an exceptionally high incidence in Asia and Africa. The number of new cases in America and Europe is rapidly increasing, making HCC a worldwide health problem. Patients with early HCC can be treated by potentially curative interventions such as tumor resection, liver transplantation, and radiofrequency ablation, but unfortunately a considerable portion of them would develop tumor recurrence, which in many cases cannot be detected early. As such, it remains imperative to develop accurate, noninvasive blood tests, which can be applied in most clinical laboratories, for the measurement of treatment responses and the surveillance for tumor recurrence. AREAS COVERED: This review article focuses on the recent discoveries of circulating proteins, DNA, microRNAs, cancer cells, and regulatory T cells as serological prognostic biomarkers for patients with HCC. These biomarkers not only have prognostic implications, but also hold promises to help physicians stratify patients for different interventions. EXPERT OPINIONS: Biomarkers will certainly serve as accurate tools in disease prognostication, changing the ways physicians stratify patients for specific therapy and monitor patients' responses toward treatment.

Stabilization of ferrochelatase via lysine residues on the carboxyl terminal extension.

There are accumulating evidences suggesting that ferrochelatase is involved in different cellular functions other than heme biosynthesis. The carboxyl-terminal extension of the enzyme may play a role associated with its stability and signaling process. Two transcript variants encoding different isoforms have been found for this ferrochelatase-coding gene. Variants encoding different isoforms have been found for this gene and were implicated in novel cellular activities including gene expression. Deficiency in ferrochelatase variants is believed to be associated with its stability. In this study, mutagenesis study of the lysine residues at 357, 360 and 365 was conducted. The relative change in activity by measurement of fluorescence intensity of the mutant enzyme revealed that K365L retained > 40% activity, while the mutant K365I retained 24% activity. Other mutants with K357S and K360Q showed little enzyme activity (< 4%). The change in the tryptophan fluorescence intensity after Guanidinium chloride-induced denaturation of the mutants indicated that the enzyme become unfolded under the assay condition. The K365L mutation suggests a structural role of tryptophan in the enzyme. The stability of the enzyme attributed to conservation of lysine residues in the C-terminal extension of the enzyme. The lysine and tryptophan residues serve as a structure determinant of ferrochelatase.

Discovery of lamin B1 and vimentin as circulating biomarkers for early hepatocellular carcinoma.

The recent advancements in proteomic technologies have reconstituted our research strategies over different type of liver diseases including hepatocellular carcinoma (HCC). Combined analyses on HCC proteome and clinicopathological data of patients have allowed identification of many promising biomarkers that can be further developed into noninvasive diagnostic assays for cancer surveillance. Capitalizing our established proteomic platform primarily based on two-dimensional polyacrylamide gel electrophoresis (2DE) and MALDI-TOF/TOF mass spectrometry, our groups have identified lamin B1 (LMNB1) and vimentin (VIM) as promising biomarkers for detection of early HCC. Protein levels of both biomarkers were significantly elevated in cancerous tissues when compared to the controls in disease-free and cirrhotic liver subjects. Further investigation of the circulating LMNB1 mRNA level in patients' blood samples by standard PCR showed 76% sensitivity and 82% specificity for detection of early HCC. In parallel, an ELISA assay for measuring circulating vimentin level in patients' serum samples could detect small HCC at 40.91% sensitivity and 87.5% specificity. The candidate biomarkers were evaluated with the diagnostic performance of α-fetoprotein (AFP) for HCC. In this article, we address the current protocols for HCC biomarker discovery, ranging from clinical sample preparation, 2DE proteomic profiling and informatics analysis, and assay development and clinical validation study. Focus is emphasized on the methods for sample preservation and low-abundance protein enrichment.

Regulators of mammalian Hippo pathway in cancer.

Hippo pathway, originally discovered in Drosophila, is responsible for organ size control. The pathway is conserved in mammals and has a significant role in restraining cancer development. Regulating the Hippo pathway thus represents a potential therapeutic approach to treat cancer, which however requires deep understanding of the targeted pathway. Despite our limited knowledge on the pathway, there are increasing discoveries of new molecules that regulate and modulate the Hippo downstream signaling particularly in various solid malignancies, from extracellular stimuli or via pathway crosstalk. Herein, we discuss the roles of newly identified and key regulators that connect with core components (MST1/2, LATS1/2, SAV1, and MOB1) and downstream effector (YAP) in the Hippo pathway having an important role in cancer development and progression. Understanding of the mammalian Hippo pathway regulation may shed new insights to allow us selecting the right oncogenic targets and designing effective drugs for cancer treatments.

Circulating miR-15b and miR-130b in serum as potential markers for detecting hepatocellular carcinoma: a retrospective cohort study.

OBJECTIVE: Serum α-fetoprotein (AFP) is the most commonly used biomarker for screening hepatocellular carcinoma (HCC) but fails to detect about half of the patients. Thus, we investigated if circulating microRNAs (miRNAs) could outperform AFP for HCC detection. DESIGN: A retrospective cohort study. SETTING: Two clinical centres in China. PARTICIPANTS: The exploration phase included 96 patients with HCC who received primary curative hepatectomy, and the validation phase included 29 hepatitis B carriers, 57 patients with HCC and 30 healthy controls. MAIN OUTCOME MEASURES: Expression of miRNAs was measured by real-time quantitative reverse transcription-PCR. Areas under receiver operating characteristic curves were used to determine the feasibility of using serum miRNA concentration as a diagnostic marker for defining HCC. A multivariate logistic regression analysis was used to evaluate performances of combined serum miRNAs. RESULTS: In the exploration phase, miRNA profiling on resected tumour/adjacent non-tumour tissues identified miR-15b, miR-21, miR-130b and miR-183 highly expressed in tumours. These miRNAs were also detectable in culture supernatants of HCC cell lines and in serum samples of patients. Remarkably, these serum miRNAs were markedly reduced after surgery, indicating the tumour-derived source of these circulating miRNAs. In a cross-centre validation study, combined miR-15b and miR-130b demonstrated as a classifier for HCC detection, yielding a receiver operating characteristic curve area of 0.98 (98.2% sensitivity and 91.5% specificity). The detection sensitivity of the classifier in a subgroup of HCCs with low AFP (<20 ng/ml) was 96.7%. The classifier also identified early-stage HCC cases that could not be detected by AFP. CONCLUSION: The combined miR-15b and miR-130b classifier is a serum biomarker with clinical value for HCC screening.

Celastrol suppresses growth and induces apoptosis of human hepatocellular carcinoma through the modulation of STAT3/JAK2 signaling cascade in vitro and in vivo.

Cumulative evidences(s) have established that the constitutive activation of STAT3 plays a pivotal role in the proliferation, survival, metastasis, and angiogenesis and thus can contribute directly to the pathogenesis of hepatocellular carcinoma (HCC). Thus, novel agents that can inhibit STAT3 activation have potential for both prevention and treatment of HCCs. The effect of celastrol on STAT3 activation, associated protein kinases, STAT3-regulated gene products, cellular proliferation, and apoptosis was investigated. The in vivo effect of celastrol on the growth of human HCC xenograft tumors in athymic nu/nu mice was also examined. We observed that celastrol inhibited both constitutive and inducible STAT3 activation, and the suppression was mediated through the inhibition of activation of upstream kinases c-Src, as well as Janus-activated kinase-1 and -2. Vanadate treatment reversed the celastrol-induced modulation of STAT3, suggesting the involvement of a tyrosine phosphatase. The inhibition of STAT3 activation by celastrol led to the suppression of various gene products involved in proliferation, survival, and angiogenesis. Celastrol also inhibited the proliferation and induced apoptosis in HCC cells. Finally, when administered intraperitoneally, celastrol inhibited STAT3 activation in tumor tissues and the growth of human HCC xenograft tumors in athymic nu/nu mice without any side effects. Overall, our results suggest for the first time that celastrol exerts its antiproliferative and proapoptotic effects through suppression of STAT3 signaling in HCC both in vitro and in vivo.

Tripterygium wilfordii bioactive compounds as anticancer and anti-inflammatory agents.

1. Bioactive compounds from medicinal plants with anticancer and anti-inflammatory effects have become key resources in drug discovery fields for the treatment of various malignancies and immunological disorders. 2. Tripterygium wilfordii (TW) is a medicinal plant that exhibits profound immunosuppressive effects and has been used in herbal regimens for the treatment of immunological diseases for thousand years in China. 3. Procedures for the isolation and characterization of TW bioactive compounds have been well established. Over the past three decades, more than 46 diterpenoids, 20 new triterpenoids, 26 alkaloids and other small molecules have been identified from TW. 4. Triptolide, celastrol and tripchlorolide are among the bioactive compounds conferring the immunosuppressive and anticancer activities of TW. Accumulated evidence suggests that the TW bioactive compounds exert their pharmacological actions by modulating the transcriptional activity of nuclear factor-κB signalling molecule. 5. Triptolide derivatives with improved water solubility have emerged as promising drug candidates. Clinical trials are being conducted on the derivatives to provide encyclopaedic knowledge on triptolide pharmacokinetics in patients.

Global regulation on microRNA in hepatitis B virus-associated hepatocellular carcinoma.

Recent work has revealed the causative links between deregulation of microRNAs (miRNAs) and cancer development. In hepatocellular carcinoma (HCC), aberrant expression of miRNAs has been observed, but the molecular mechanisms that contribute to such changes remains to be elucidated. Here, we reported the analysis of miRNA expression in 94 pairs of tumor and adjacent nontumor tissues from HBV-associated HCC in Chinese patients. We found miRNAs were aberrantly expressed in HCC tissues. To investigate the cause of such deregulation, we detected changes in DNA copy number by measuring locus-specific hybridization intensity, and found changes in expression of several miRNAs are correlated with genomic amplification or deletion. For example, the genomic regions of miR-30d and miR-151 were amplified in ∼50% of HCC tumor tissues, and the expressions of these miRNAs are significantly correlated with DNA copy number. We also employed cDNA microarray data, and provide evidence that key regulators of the miRNA biosynthetic pathway, including DROSHA, DGCR8, AGO1, and AGO2, are frequently overexpressed in HCC. This study provides molecular clues that may contribute to the global changes of miRNA expression in HCC.

microRNA-122 as a regulator of mitochondrial metabolic gene network in hepatocellular carcinoma.

Tumorigenesis involves multistep genetic alterations. To elucidate the microRNA (miRNA)-gene interaction network in carcinogenesis, we examined their genome-wide expression profiles in 96 pairs of tumor/non-tumor tissues from hepatocellular carcinoma (HCC). Comprehensive analysis of the coordinate expression of miRNAs and mRNAs reveals that miR-122 is under-expressed in HCC and that increased expression of miR-122 seed-matched genes leads to a loss of mitochondrial metabolic function. Furthermore, the miR-122 secondary targets, which decrease in expression, are good prognostic markers for HCC. Transcriptome profiling data from additional 180 HCC and 40 liver cirrhotic patients in the same cohort were used to confirm the anti-correlation of miR-122 primary and secondary target gene sets. The HCC findings can be recapitulated in mouse liver by silencing miR-122 with antagomir treatment followed by gene-expression microarray analysis. In vitro miR-122 data further provided a direct link between induction of miR-122-controlled genes and impairment of mitochondrial metabolism. In conclusion, miR-122 regulates mitochondrial metabolism and its loss may be detrimental to sustaining critical liver function and contribute to morbidity and mortality of liver cancer patients.

Prophylactic uses of integrin CD18-betaA peptide in a murine polymicrobial peritonitis model.

AIM: To evaluate the prophylactic properties of integrin CD18-betaA peptide in a murine model of abdominal polymicrobial peritonitis and sepsis. METHODS: Bacterial sepsis was induced in Institute of Cancer Research (ICR) mice by cecal ligation and puncture (CLP) surgery. Inflicted mice were then injected with either sterile saline or CD18-betaA peptide intraperitoneally at 2 h after surgery, and were sacrificed at 12 and 24 h after surgery. Blood samples were immediately collected, and analyzed for endotoxin activity and tumor necrosis factor (TNF)-alpha and interleukin (IL)-6. Lungs and liver were studied for CD45+ leukocyte and CD3 mRNA content. Pulmonary expression of intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM) and E-selectin was also determined. RESULTS: Intraperitoneal injection of CD18-betaA peptide significantly suppressed circulating endotoxin activity (P < 0.01) at 24 h, as well as serum levels of TNF-alpha (P < 0.05 at 12 and 24 h) and IL-6 (P < 0.01 at 12 h, P < 0.05 at 24 h) in CLP-inflicted mice. CD18-betaA peptide also abrogated leukocyte infiltration into liver and lungs as unveiled by reduced CD45+ leukocyte and CD3 mRNA contents. Furthermore, the peptide significantly reduced pulmonary expression of VCAM (P < 0.01 at 12 h, P < 0.001 at 24 h), E-selectin (P < 0.01 at 12 and 24 h), and ICAM-1 (P < 0.01 at 12 h, P < 0.001 at 24 h). These actions of CD18-betaA peptide collectively protected septic mice against lethality (P < 0.01). CONCLUSION: CD18-betaA peptide is a potent endotoxin antagonist that can protect surgical patients against sepsis-associated lethality.

HNF1α and CDX2 transcriptional factors bind to cadherin-17 (CDH17) gene promoter and modulate its expression in hepatocellular carcinoma.

Cadherin-17 (CDH17) belongs to the cell adhesion cadherin family with a prominent role in tumorigenesis. It is highly expressed in human hepatocellular carcinoma (HCC) and is proposed to be a biomarker and therapeutic molecule for liver malignancy. The present study aims to identify the transcription factors which interact and regulate CDH17 promoter activity that might contribute to the up-regulation of CDH17 gene in human HCC. A 1-kb upstream sequence of CDH17 gene was cloned and the promoter activity was studied by luciferase reporter assay. By bioinformatics analysis, deletion and mutation assays, and chromatin immunoprecipitation studies, we identified hepatic nuclear factor 1α (HNF1α) and caudal-related homeobox 2 (CDX2) binding sites at the proximal promoter region which modulate the CDH17 promoter activities in two HCC cell lines (Hep3B and MHCC97L). A consistent down-regulation of CDH17 and the two transcriptional activators (HNF1α and CDX2) expression was found in the liver of mouse during development, as well as in human liver cancer cells with less metastatic potential. Suppression of HNF1α and CDX2 expression by small interfering RNA (siRNA) significantly down-regulated expressions of CDH17 and its downstream target cyclin D1 and the viability of HCC cells in vitro. In summary, we identified the minimal promoter region of CDH17 that is regulated by HNF1α and CDX2 transcriptional factors. The present findings enhance our understanding on the regulatory mechanisms of CDH17 oncogene in HCC, and may shed new insights into targeting CDH17 expression as potential therapeutic intervention for cancer treatment.

Enhanced detection of early hepatocellular carcinoma by serum SELDI-TOF proteomic signature combined with alpha-fetoprotein marker.

BACKGROUND: Biomarkers for accurate diagnosis of early hepatocellular carcinoma (HCC) are limited in number and clinical validation. We applied SELDI-TOF-MS ProteinChip technology to identify serum profile for distinguishing HCC and liver cirrhosis (LC) and to compare the accuracy of SELDI-TOF-MS profile and alpha-fetoprotein (AFP) level in HCC diagnosis. PATIENTS AND METHODS: Serum samples were obtained from 120 HCC and 120 LC patients for biomarker discovery and validation studies. ProteinChip technology was employed for generating SELDI-TOF proteomic features and analyzing serum proteins/peptides. RESULTS: A diagnostic model was established by CART algorithm, which is based on 5 proteomic peaks with m/z values at 3324, 3994, 4665, 4795, and 5152. In the training set, the CART algorithm could differentiate HCC from LC subjects with a sensitivity and specificity of 98% and 95%, respectively. The results were assessed in blind validation using separate cohorts of 60 HCC and 60 LC patients, with an accuracy of 83% for HCC and 92% for LC patients. The diagnostic odd ratio (DOR) indicated that SELDI-TOF proteomic signature could achieve better diagnostic performance than serum AFP level at a cutoff of 20 ng/mL (AFP(20)) (92.72 vs 9.11), particularly superior for early-stage HCC (87% vs 54%). Importantly, a combined use of both tests could enhance the detection of HCC (sensitivity, 95%; specificity, 98%; DOR, 931). CONCLUSION: Serum SELDI-TOF proteomic signature, alone or in combination with AFP marker, promises to be a good tool for early diagnosis and/screening of HCC in at-risk population with liver cirrhosis.

Endotoxin-neutralizing peptides as gram-negative sepsis therapeutics.

Bacterial endotoxin [e.g. lipopolysaccharide (LPS)] can trigger systemic hyper-inflammatory that subsequently leads to multiple organ failure and lethality (gram-negative sepsis). This paper describes the development of endotoxin-neutralizing peptides that potentially treat sepsis. These peptides have been derived from bactericidal/permeability-increasing protein (BPIP), anti-microbial peptides, and leukocyte CD18 antigen and some of these peptides have been tested in clinical studies.