Michael Hsiao

MicroRNA-122 plays a critical role in liver homeostasis and hepatocarcinogenesis

MicroRNA-122 (miR-122), which accounts for 70% of the livers total miRNAs, plays a pivotal role in the liver. However, its intrinsic physiological roles remain largely undetermined. We demonstrated that mice lacking the gene encoding miR-122a (Mir122a) are viable but develop temporally controlled steatohepatitis, fibrosis, and hepatocellular carcinoma (HCC). These mice exhibited a striking disparity in HCC incidence based on sex, with a male-to-female ratio of 3.9:1, which recapitulates the disease incidence in humans. Impaired expression of microsomal triglyceride transfer protein (MTTP) contributed to steatosis, which was reversed by in vivo restoration of Mttp expression. We found that hepatic fibrosis onset can be partially attributed to the action of a miR-122a target, the Klf6 transcript. In addition, Mir122a(-/-) livers exhibited disruptions in a range of pathways, many of which closely resemble the disruptions found in human HCC. Importantly, the reexpression of miR-122a reduced disease manifestation and tumor incidence in Mir122a(-/-) mice. This study demonstrates that mice with a targeted deletion of the Mir122a gene possess several key phenotypes of human liver diseases, which provides a rationale for the development of a unique therapy for the treatment of chronic liver disease and HCC.

MicroRNA‐122, a tumor suppressor microRNA that regulates intrahepatic metastasis of hepatocellular carcinoma

MicroRNAs (miRNAs), which are inhibitors of gene expression, participate in diverse biological functions and in carcinogenesis. In this study, we show that liver‐specific microRNA‐122 (miR‐122) is significantly down‐regulated in liver cancers with intrahepatic metastastasis and negatively regulates tumorigenesis. Restoration of miR‐122 in metastatic Mahlavu and SK‐HEP‐1 cells significantly reduced in vitro migration, invasion, and anchorage‐independent growth as well as in vivo tumorigenesis, angiogenesis, and intrahepatic metastasis in an orthotopic liver cancer model. Because an inverse expression pattern is often present between an miRNA and its target genes, we used a computational approach and identified multiple miR‐122 candidate target genes from two independent expression microarray datasets. Thirty‐two target genes were empirically verified, and this group of genes was enriched with genes regulating cell movement, cell morphology, cell‐cell signaling, and transcription. We further showed that one of the miR‐122 targets, ADAM17 (a disintegrin and metalloprotease 17) is involved in metastasis. Silencing of ADAM17 resulted in a dramatic reduction of in vitro migration, invasion, in vivo tumorigenesis, angiogenesis, and local invasion in the livers of nude mice, which is similar to that which occurs with the restoration of miR‐122. Conclusion: Our study suggests that miR‐122, a tumor suppressor microRNA affecting hepatocellular carcinoma intrahepatic metastasis by angiogenesis suppression, exerts some of its action via regulation of ADAM17. Restoration of miR‐122 has a far‐reaching effect on the cell. Using the concomitant down‐regulation of its targets, including ADAM17, a rational therapeutic strategy based on miR‐122 may prove to be beneficial for patients with hepatocellular carcinoma. (HEPATOLOGY 2009.)

H3K9 histone methyltransferase G9a promotes lung cancer invasion and metastasis by silencing the cell adhesion molecule Ep-CAM

G9a is a mammalian histone methyltransferase that contributes to the epigenetic silencing of tumor suppressor genes. Emerging evidence suggests that G9a is required to maintain the malignant phenotype, but the role of G9a function in mediating tumor metastasis has not been explored. Here, we show that G9a is expressed in aggressive lung cancer cells, and its elevated expression correlates with poor prognosis. RNAi-mediated knockdown of G9a in highly invasive lung cancer cells inhibited cell migration and invasion in vitro and metastasis in vivo. Conversely, ectopic G9a expression in weakly invasive lung cancer cells increased motility and metastasis. Mechanistic investigations suggested that repression of the cell adhesion molecule Ep-CAM mediated the effects of G9a. First, RNAi-mediated knockdown of Ep-CAM partially relieved metastasis suppression imposed by G9a suppression. Second, an inverse correlation between G9a and Ep-CAM expression existed in primary lung cancer. Third, Ep-CAM repression was associated with promoter methylation and an enrichment for dimethylated histone H3K9. G9a knockdown reduced the levels of H3K9 dimethylation and decreased the recruitment of the transcriptional cofactors HP1, DNMT1, and HDAC1 to the Ep-CAM promoter. Our findings establish a functional contribution of G9a overexpression with concomitant dysregulation of epigenetic pathways in lung cancer progression.

Enhancement of cell radiation sensitivity by pegylated gold nanoparticles

Biocompatible Au nanoparticles with surfaces modified by PEG (polyethylene glycol) were developed in view of possible applications for the enhancement of radiotherapy. Such nanoparticles exhibit preferential deposition at tumor sites due to the enhanced permeation and retention (EPR) effect. Here, we systematically studied their effects on EMT-6 and CT26 cell survival rates during irradiation for a dose up to 10 Gy with a commercial biological irradiator (E(average) = 73 keV), a Cu-Kalpha(1) x-ray source (8.048 keV), a monochromatized synchrotron source (6.5 keV), a radio-oncology linear accelerator (6 MeV) and a proton source (3 MeV). The percentage of surviving cells after irradiation was found to decrease by approximately 2-45% in the presence of PEG-Au nanoparticles ([Au] = 400, 500 or 1000 microM). The cell survival rates decreased as a function of the dose for all sources and nanoparticle concentrations. These results could open the way to more effective cancer irradiation therapies by using nanoparticles with optimized surface treatment. Difficulties in applying MTT assays were also brought to light, showing that this approach is not suitable for radiobiology.

MicroRNA-373 (miR-373) post-transcriptionally regulates large tumor suppressor, homolog 2 (LATS2) and stimulates proliferation in human esophageal cancer.

LATS2 is a member of the LATS tumor suppressor family. It has been implicated in regulation of the cell cycle and apoptosis. Frequent loss of heterozygosity (LOH) of LATS2 has been reported in human esophageal cancer. But, the LATS2 gene expression and its regulatory mechanism in esophageal cancer remain unclear. The present study has shown that LATS2 protein expression was mediated by miR-373 at the post-transcriptional level and inversely correlated with miR-373 amounts in esophageal cancer cell lines. Furthermore, we demonstrated that the direct inhibition of LATS2 protein was mediated by miR-373 and manipulated the expression of miR-373 to affect esophageal cancer cells growth. Moreover, this correlation was supported by data collected ex vivo, in which esophageal cancer tissues from esophageal squamous cell carcinoma (ESCC) patients were analyzed. Finally, by miRNA microarray analysis, four miRNAs including miR-373 were over-expressed in ESCC samples. Our findings reveal that miR-373 would be a potential oncogene and it participates in the carcinogenesis of human esophageal cancer by suppressing LATS2 expression.

MicroRNA-330 acts as tumor suppressor and induces apoptosis of prostate cancer cells through E2F1-mediated suppression of Akt phosphorylation

MicroRNAs (miRNAs) make up a novel class of gene regulators; they function as oncogenes or tumor suppressors by targeting tumor-suppressor genes or oncogenes. A recent study that analysed a large number of human cancer cell lines showed that miR-330 is a potential tumor-suppressor gene. However, the function and molecular mechanism of miR-330 in determining the aggressiveness of human prostate cancer has not been studied. Here, we show that miR-330 is significantly lower expressed in human prostate cancer cell lines than in nontumorigenic prostate epithelial cells. Bioinformatics analyses reveal a conserved target site for miR-330 in the 3′-untranslated region (UTR) of E2F1 at nucleotides 1018–1024. MiR-330 significantly suppressed the activity of a luciferase reporter containing the E2F1-3′-UTR in the cells. This activity could be abolished with the transfection of anti-miR-330 or mutated E2F1-3′-UTR. In addition, the expression level of miR-330 and E2F1 was inversely correlated in cell lines and prostate cancer specimens. After overexpressing of miR-330 in PC-3 cells, cell growth was suppressed by reducing E2F1-mediated Akt phosphorylation and thereby inducing apoptosis. Collectively, this is the first study to show that E2F1 is negatively regulated by miR-330 and also show that miR-330 induces apoptosis in prostate cancer cells through E2F1-mediated suppression of Akt phosphorylation.

Biocompatibility of Fe(3)O(4) nanoparticles evaluated by in vitro cytotoxicity assays using normal, glia and breast cancer cells.

In order to reveal the biocompatibility of Fe(3)O(4) nanoparticles and bipolar surfactant tetramethylammonium 11-aminoundecanoate cytotoxicity tests were performed as a function of concentration from low (0.1 microg ml(-1)) to higher concentration (100 microg ml(-1)) using various human glia, human breast cancer and normal cell lines. Cytotoxicity tests for human glia (D54MG, G9T, SF126, U87, U251, U373), human breast cancer (MB157, SKBR3, T47D) and normal (H184B5F5/M10, WI-38, SVGp12) cell lines exhibited almost nontoxicity and reveal biocompatibility of Fe(3)O(4) nanoparticles in the concentration range of 0.1-10 microg ml(-1), while accountable cytotoxicity can be seen at 100 microg ml(-1). The results of our studies suggest that Fe(3)O(4) nanoparticles coated with bipolar surfactant tetramethylammonium 11-aminoundecanoate are biocompatible and promising for bio-applications such as drug delivery, magnetic resonance imaging and magnetic hyperthermia.

Identification of SOX4 target genes using phylogenetic footprinting-based prediction from expression microarrays suggests that overexpression of SOX4 potentiates metastasis in hepatocellular carcinoma

A comprehensive microarray analysis of hepatocellular carcinoma (HCC) revealed distinct synexpression patterns during intrahepatic metastasis. Recent evidence has demonstrated that synexpression group member genes are likely to be regulated by master control gene(s). Here we investigate the functions and gene regulation of the transcription factor SOX4 in intrahepatic metastatic HCC. SOX4 is important in tumor metastasis as RNAi knockdown reduces tumor cell migration, invasion, in vivo tumorigenesis and metastasis. A multifaceted approach integrating gene profiling, binding site computation and empirical verification by chromatin immunoprecipitation and gene ablation refined the consensus SOX4 binding motif and identified 32 binding loci in 31 genes with high confidence. RNAi knockdown of two SOX4 target genes, neuropilin 1 and semaphorin 3C, drastically reduced cell migration activity in HCC cell lines suggesting that SOX4 exerts some of its action via regulation of these two downstream targets. The discovery of 31 previously unidentified targets expands our knowledge of how SOX4 modulates HCC progression and implies a range of novel SOX4 functions. This integrated approach sets a paradigm whereby a subset of member genes from a synexpression group can be regulated by one master control gene and this is exemplified by SOX4 and advanced HCC.

IL-6 Trans-Signaling in Formation and Progression of Malignant Ascites in Ovarian Cancer

Classic signaling by the proinflammatory cytokine interleukin 6 (IL-6) involves its binding to target cells that express the membrane-bound IL-6 receptor α. However, an alternate signaling pathway exists in which soluble IL-6 receptor (sIL-6Rα) can bind IL-6 and activate target cells that lack mIL-6Rα, such as endothelial cells. This alternate pathway, also termed trans-signaling, serves as the major IL-6 signaling pathway in various pathologic proinflammatory conditions including cancer. Here we report that sIL-6Rα is elevated in malignant ascites from ovarian cancer patients, where it is associated with poor prognosis. IL-6 trans-signaling on endothelial cells prevented chemotherapy-induced apoptosis, induced endothelial hyperpermeability, and increased transendothelial migration of ovarian cancer cells. Selective blockade of the MAPK pathway with ERK inhibitor PD98059 reduced IL-6/sIL-6Rα-mediated endothelial hyperpermeability. ERK activation by the IL-6/sIL-6Rα complex increased endothelial integrity via Src kinase activation and Y685 phosphorylation of VE-cadherin. Selective targeting of IL-6 trans-signaling in vivo reduced ascites formation and enhanced the taxane sensitivity of intraperitoneal human ovarian tumor xenografts in mice. Collectively, our results show that increased levels of sIL-6Rα found in ovarian cancer ascites drive IL-6 trans-signaling on endothelial cells, thereby contributing to cancer progression. Selective blockade of IL-6 trans-signaling may offer a promising therapeutic strategy to improve the management of patients with advanced ovarian cancer.

Seedless, silver-induced synthesis of star-shaped gold/silver bimetallic nanoparticles as high efficiency photothermal therapy reagent

This work demonstrates a simple method for synthesizing a shape-controllable bimetallic gold/silver nanostructured material. Spiky star-shaped gold/silver nanoparticles are obtained by mixing HAuCl4, AgNO3 and ascorbic acid with shaking for 20 s. The wide range of star shapes and irregular quasi-spherical nanoparticles is tailored by tuning the ratio of metal precursors. The wavelengths absorbed by the nanoparticles can be tuned from visible light to near infrared by controlling their shape. To maintain the morphology of the nanoparticles, enhance their thermal stability and support their application in biological systems, modified chitosan was utilized for the properties and to keep the material well dispersed in solution in deionized water. The moderate concentration of modified chitosan capped bimetallic star-shaped nanoparticles not only ensured non-toxicity to normal cells and cancer cells, but also promoted high efficiency photothermal ablation of cancer cells. Ultimately, this nanotechnology-driven assay has huge potential for application in rapid synthesis, tunable absorption and non-cytotoxic photothermal therapy for the effective treatment of cancer.