Kam M. Hui

MicroRNA-216a/217-induced epithelial-mesenchymal transition targets PTEN and SMAD7 to promote drug resistance and recurrence of liver cancer

Tumor recurrence and metastases are the major obstacles to improving the prognosis of patients with hepatocellular carcinoma (HCC). To identify novel risk factors associated with HCC recurrence and metastases, we have established a panel of recurrence‐associated microRNAs (miRNAs) by comparing miRNA expression in recurrent and nonrecurrent human HCC tissue samples using microarrays (recurrence is defined as recurrent disease occurring within a 2‐year time point of the original treatment). Among the panel, expression of the miR‐216a/217 cluster was consistently and significantly up‐regulated in HCC tissue samples and cell lines associated with early tumor recurrence, poor disease‐free survival, and an epithelial‐mesenchymal transition (EMT) phenotype. Stable overexpression of miR‐216a/217‐induced EMT increased the stem‐like cell population, migration, and metastatic ability of epithelial HCC cells. Phosphatase and tensin homolog (PTEN) and mothers against decapentaplegic homolog 7 (SMAD7) were subsequently identified as two functional targets of miR‐216a/217, and both PTEN and SMAD7 were down‐regulated in HCC. Ectopic expression of PTEN or SMAD7 partially rescued miR‐216a/217‐mediated EMT, cell migration, and stem‐like properties of HCC cells. Previously, SMAD7 was shown to be a transforming growth factor beta (TGF‐β) type 1 receptor antagonist. Here, we further demonstrated that overexpression of miR‐216a/217 acted as a positive feedback regulator for the TGF‐β pathway and the canonical pathway involved in the activation of phosphoinositide 3‐kinase/protein kinase K (PI3K/Akt) signaling in HCC cells. Additionally, activation of the TGF‐β‐ and PI3K/Akt‐signaling pathways in HCC cells resulted in an acquired resistance to sorafenib, whereas blocking activation of the TGF‐β pathway overcame miR‐216a/217‐induced sorafenib resistance and prevented tumor metastases in HCC. Conclusion: Overexpression of miR‐216a/217 activates the PI3K/Akt and TGF‐β pathways by targeting PTEN and SMAD7, contributing to hepatocarcinogenesis and tumor recurrence in HCC. (Hepatology 2013;58:629–641)

Potential role of signal transducer and activator of transcription (STAT)3 signaling pathway in inflammation, survival, proliferation and invasion of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies, and is also the fourth most common cancer worldwide with around 700,000 new cases each year. Currently, first line chemotherapeutic drugs used for HCC include fluorouracil, cisplatin, doxorubicin, paclitaxel and mitomycin, but most of these are non-selective cytotoxic molecules with significant side effects. Sorafenib is the only approved targeted therapy by the U.S. Food and Drug Administration for HCC treatment, but patients suffer from various kinds of adverse effects, including hypertension. The signal-transducer-and-activator-of-transcription 3 (STAT3) protein, one of the members of STATs transcription factor family, has been implicated in signal transduction by different cytokines, growth factors and oncogenes. In normal cells, STAT3 activation is tightly controlled to prevent dysregulated gene transcription, whereas constitutively activated STAT3 plays an important role in tumorigenesis through the upregulation of genes involved in anti-apoptosis, proliferation and angiogenesis. Thus, pharmacologically safe and effective agents that can block STAT3 activation have the potential both for the prevention and treatment of HCC. In the present review, we discuss the possible role of STAT3 signaling cascade and its interacting partners in the initiation of HCC and also analyze the role of various STAT3 regulated genes in HCC progression, inflammation, survival, invasion and angiogenesis.

Paper-Based Microfluidic Electrochemical Immunodevice Integrated with Nanobioprobes onto Graphene Film for Ultrasensitive Multiplexed Detection of Cancer Biomarkers

To the best of our knowledge, this was the first report on the integration of a signal amplification strategy into a microfluidic paper-based electrochemical immunodevice for the multiplexed measurement of cancer biomarkers. Signal amplification was achieved through the use of graphene to modify the immunodevice surface to accelerate the electron transfer and the use of silica nanoparticles as a tracing tag to label the signal antibodies. Accurate, rapid, simple, and inexpensive point-of-care electrochemical immunoassays were demonstrated using a photoresist-patterned microfluidic paper-based analytical device (μPAD). Using the horseradish peroxidase (HRP)-O-phenylenediamine-H2O2 electrochemical detection system, the potential clinical applicability of this immunodevice was demonstrated through its ability to identify four candidate cancer biomarkers in serum samples from cancer patients. The novel signal-amplified strategy proposed in this report greatly enhanced the sensitivity of the detection of cancer biomarkers. In addition, the electrochemical immunodevice exhibited good stability, reproducibility, and accuracy and thus had potential applications in clinical diagnostics.

MiR-214 Targets β-Catenin Pathway to Suppress Invasion, Stem-Like Traits and Recurrence of Human Hepatocellular Carcinoma

The down-regulation of miR-214 has previously been observed in human hepatocellular carcinoma (HCC). Here, we demonstrated the down-regulation of miR-214 is associated with cell invasion, stem-like traits and early recurrence of HCC. Firstly, we validated the suppression of miR-214 in human HCC by real-time quantitative RT-PCR (qRT-PCR) in 20 paired tumor and non-tumor liver tissues of HCC patients and 10 histologically normal liver tissues from colorectal cancer patients with liver metastases. Further qRT-PCR analysis of 50 HCC tissues from an independent cohort of HCC patients of whom 29 with early recurrent disease (<2 years) and 21 with late recurrent disease demonstrated that the suppression of miR-214 was significantly more suppressed in samples from HCC patients with early recurrent disease compared those from patients with no recurrence. Re-expression of miR-214 significantly suppressed the growth of HCC cells in vitro and reduced their tumorigenicity in vivo. The enhancer of zeste homologue 2 (EZH2) and β-catenin (CTNNB1) was identified as two potential direct downstream targets of miR-214 through bioinformatics analysis and experimentally validated the miRNA-target interactions with a dual-firefly luciferase reporter assay. In corroborate with this, both EZH2 and CTNNB1 are found to be significantly overexpressed in human HCC biopsies. Since EZH2 can regulate CTNNB1, CTNNB1 can also be an indirect target of miR-214 through EZH2. Silencing EZH2 or CTNNB1 expression suppressed the growth and invasion of HCC cells and induced E-cadherin (CDH1), known to inhibit cell invasion and metastasis. Furthermore, the silencing of miR-214 or overexpression of EZH2 increased EpCAM+ stem-like cells through the activation of CTNNB1. Interestingly, the up-regulation of EZH2, CTNNB1 and the down-regulation of CDH1 in HCC patients correlated with early recurrent disease and can be an independent predictor of poor survival. Therefore, miR-214 can directly or indirectly target CTNNB1 to modulate the β-catenin signaling pathway in HCC.

Matrix metalloproteinase 1 is necessary for the migration of human bone marrow-derived mesenchymal stem cells toward human glioma.

Human mesenchymal stem cells (MSCs) have increasingly been used as cellular vectors for the delivery of therapeutic genes to tumors. However, the precise mechanism of mobilization remains poorly defined. In this study, MSCs that expressed similar cell surface markers and exhibited multilineage differentiation potentials were isolated from various donors. Interestingly, different MSC isolates displayed differential migration ability toward human glioma cells. We hypothesized that distinct molecular signals may be involved in the varied tumor tropisms exhibited by different MSC isolates. To test this hypothesis, gene expression profiles of tumor‐trophic MSCs were compared with those of non–tumor‐trophic MSCs. Among the various differentially regulated genes, matrix metalloproteinase one (MMP1) gene expression and its protein activities were enhanced by 27‐fold and 21‐fold, respectively, in highly migrating MSCs compared with poorly migrating MSCs. By contrast, there was no change in the transcriptional levels of other MMPs. Functional inactivation of MMP1 abrogated the migratory potential of MSCs toward glioma‐conditioned medium. Conversely, the nonmigratory phenotype of poorly migrating MSC could be rescued in the presence of either recombinant MMP1 or conditioned medium from the highly migrating MSCs. Ectopic expression of MMP1 in these poorly migrating cells also rendered the cells responsive to the signaling cues from the glioma cells in vivo. However, blocking the interaction of MMP1 and its cognate receptor PAR1 effectively diminished the migratory ability of MSCs. Taken together, this study provides, for the first time, supporting evidence that MMP1 is critically involved in the migration capacity of MSCs, acting through the MMP1/PAR1 axis. STEM CELLS 2009;27:1366–1375

Diosgenin, a steroidal saponin, inhibits STAT3 signaling pathway leading to suppression of proliferation and chemosensitization of human hepatocellular carcinoma cells

Constitutive activation of STAT3 has been shown in several human cancers and transformed cell lines including hepatocellular carcinoma (HCC). In the present report, we investigated whether diosgenin, a steroidal saponin isolated from fenugreek can modulate the STAT3 signaling pathway. We found that diosgenin inhibited both constitutive and inducible activation of STAT3 with no effect on STAT5. The activation of c-Src, JAK1 and JAK2 implicated in STAT3 activation, were also suppressed by this saponin. Pervanadate reversed the diosgenin-induced downregulation of STAT3, suggesting the involvement of a protein tyrosine phosphatase. Indeed, we found that diosgenin can induce the expression of Src homology 2 phosphatase 2 (SH-PTP2) that correlated with downregulation of constitutive STAT3 activation. Diosgenin also downregulated the expression of various STAT3-regulated gene products, inhibited proliferation and potentiated the apoptotic effects of paclitaxel and doxorubicin. Overall, these results suggest that diosgenin is a novel blocker of the STAT3 activation pathway, with a potential role in the treatment of HCC and other cancers.

A paper-based microfluidic electrochemical immunodevice integrated with amplification-by-polymerization for the ultrasensitive multiplexed detection of cancer biomarkers

A novel signal amplification strategy for ultrasensitive multiplexed detection of cancer biomarkers using a paper-based microfluidic electrochemical immunodevice is described. Specifically, a controlled radical polymerization reaction is triggered after the capture of target molecules on the immunodevice surface. Growth of long chain polymeric materials provides numerous sites for subsequent horseradish peroxidase (HRP) coupling, which in turn significantly enhances electrochemical signal output. The signal was further amplified through the use of graphene to modify the immunodevice surface to accelerate the electron transfer. Activators generated electron transfer for atom transfer radical polymerization (AGET ATRP) was used in this study for its high efficiency in polymer grafting and better tolerance toward oxygen in air. Glycidyl methacrylate (GMA) was examined to provide excess epoxy groups for HRP coupling. In the electrochemical immunodevice, eight carbon working electrodes, as well as their conductive pads, were screen-printed on a piece of square paper, and the same Ag/AgCl reference and carbon counter electrodes were shared with another piece of square paper via stacking. Using the HRP-O-phenylenediamine-H2O2 electrochemical detection system, four cancer biomarkers: carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), cancer antigen 125 (CA125), and carbohydrate antigen 153 (CA153) were detected. A limit of detection of 0.01, 0.01, 0.05 and 0.05 ng mL(-1) was demonstrated, respectively. The results show that the proposed strategy offers great promises in providing a sensitive and cost-effective solution for biosensing applications.

Generation of cytokine‐induced killer cells from leukaemic samples with in vitro cytotoxicity against autologous and allogeneic leukaemic blasts

Summary. Cytokine‐induced killer (CIK) cells are CD3+CD56+ non‐major histocompatibility complex (MHC)‐restricted immune effector cells. The present report demonstrates that it was possible to expand CIK cells obtained at diagnosis from patients with acute leukaemia. The percentage of CD3+CD56+ CIK cells generated following culture ranged between 7·6% and 65% (median of 35·3%) and these cells were able to kill the human natural killer target K562 cells. Although the same effector cells were able to lyse autologous acute myeloid leukaemia (AML) target cells, they were not able to lyse autologous acute lymphoblastic leukaemia target cells. Pre‐absorption of the CIK effector cells by K562 cells did not completely abrogate the cytotoxicity of CIK cells against autologous blasts in 9 out of 12 samples tested. Moreover, it was observed that the cytotoxicity generated by the CIK effector cells against allogeneic leukaemic blasts was similar to that against autologous blasts. The present study suggests the potential application of CIK cells in the immunotherapy of AML, either in minimal disease state, as donor lymphocyte infusion in relapse post allogeneic transplant, or in cases of chemotherapy refractory leukaemia.

Phase I study of immunotherapy of cutaneous metastases of human carcinoma using allogeneic and xenogeneic MHC DNA-liposome complexes.

The generation of strong tumor-specific immunity by in situ gene therapy is an attractive approach for the eradication of human cancer lesions. The objectives of this study were to examine the toxicities of employing the human HLA-A2, HLA-B13 and the murine H-2Kk genes to generate tumor regression in patients with different cancer types via DC-Chol/DOPE cationic liposomes. The study was composed of two phaseI/II trials involving a total of 19 late-stage cancer patients. The patients were given four weekly injections of a DNA–liposome mixture directly into a cutaneous nodule. These procedures resulted in no significant clinical side-effects. The HLA-A2 gene gave the highest level of expression in situ. Although all patients treated had progressive systemic disease and eventually succumbed to their disease, strong local responses were generated in the treated nodules. Of the eight patients whose cutaneous nodules received HLA-A2 DNA, two completely regressed while four tumor nodules gave a partial local response. All but one of the patients who received HLA-A2–liposome mixtures and had a subsequent local response were either cervical or ovarian carcinoma patients. This local response, seen in a group of patients who had relapsed stage IV systemic metastatic disease and were refractory to all available therapies, demonstrates the generation of a strong local immune response following our in situ gene therapy protocol. Further studies to investigate the use of HLA-A2 DC-Chol/DOPE cationic liposomes for immunotherapy of cervical and ovarian cancers are warranted.

Upregulation of Rac GTPase-Activating Protein 1 Is Significantly Associated with the Early Recurrence of Human Hepatocellular Carcinoma

Purpose: To assess the significance of Rac GTPase-activating protein 1 (RACGAP1) expression in identifying HBV-positive human hepatocellular carcinoma (HCC) patients who are at high risk for recurrent disease. Experimental Design: The prognostic significance of RACGAP1 was compared with clinicopathologic parameters available at diagnosis using multivariate and log-rank test. RACGAP1 expression and outcome in recurrence was compared between 35 patients with recurrence and 41 patients without recurrence using Kaplan–Meier analysis. RACGAP1-targeted molecules and pathways were identified and characterized by inhibition with siRNA duplexes. Results: Kaplan–Meier analysis showed that the level of RACGAP1 expression is sufficient to predict the early recurrence of HCC: high RACGAP1 expression correlates with high risk of postresection recurrent HCC (P < 0.0005). Silencing of RACGAP1 in Hep3B and MHCC97-H HCC cells with high endogenous RACGAP1 expression inhibited cell migration and invasion. Using Ingenuity Pathway Analysis, the target molecules silenced in the RACGAP1 interactome were mostly genes related to the mitotic roles of the polo-like kinases. These included PRC1, AURKB, CDC2, ECT2, KIF23, PAK1, and PPP2R5E. In providing clinical corroboration of these results, when expression of these transcripts was analyzed in an expression database that we have established previously for HBV-positive HCC patients, these genes was mostly upregulated in patients who exhibited early recurrent disease and hence provided important corroboration of these results. Conclusions: siRNA-silencing RACGAP1 mainly targeted genes in an interactome clinically relevant to early HCC recurrence. Besides being an independent informative prognostic biomarker, RACGAP1 could also be a potential molecular target for designing therapeutic strategies for HCC. Clin Cancer Res; 17(18); 6040–51. ©2011 AACR.