Thu Le Trinh

Iron regulator hepcidin exhibits antiviral activity against hepatitis C virus.

Hepatitis C viral infection affects 170 million people worldwide. It causes serious chronic liver diseases. HCV infection has been implicated in iron accumulation in the liver and iron overload has been shown to be a potential cofactor for HCV associated hepatocellular carcinoma progression. The underlying mechanisms are not understood. Human hepcidin, a 25 amino acid peptide mainly produced by hepatocytes, is a key regulator of iron metabolism. Alteration of hepcidin expression levels has been reported in the setting of chronic HCV infection and hepatocellular carcinoma. In this study, we aim to examine the interactions between HCV infection and hepcidin expression in liver cells. We found that hepcidin expression was suppressed in HCV infected cells. The suppressive effect appears to be regulated by histone acetylation but not DNA methylation. Moreover, we found that hepcidin had a direct antiviral activity against HCV replication in cell culture. The antiviral effect is associated with STAT3 activation. In conclusion, hepcidin can induce intracellular antiviral state while HCV has a strategy to suppress hepcidin expression. This may be a novel mechanism by which HCV circumvents hepatic innate antiviral defense.

Connective tissue growth factor and integrin αvβ6: A new pair of regulators critical for ductular reaction and biliary fibrosis in mice

Connective tissue growth factor (CTGF) is a matricellular protein that mediates cell‐matrix interaction through various subtypes of integrin receptors. This study investigated the role of CTGF and integrin αvβ6 in hepatic progenitor/oval cell activation, which often occurs in the form of ductular reactions (DRs) when hepatocyte proliferation is inhibited during severe liver injury. CTGF and integrin αvβ6 proteins were highly expressed in DRs of human cirrhotic livers and cholangiocarcinoma. Confocal microscopy analysis of livers from Ctgf promoter‐driven green fluorescent protein reporter mice suggested that oval cells and cholangiocytes were the main sources of CTGF and integrin αvβ6 during liver injury induced by 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC). Deletion of exon 4 of the Ctgf gene using tamoxifen‐inducible Cre‐loxP system down‐regulated integrin αvβ6 in DDC‐damaged livers of knockout mice. Ctgf deficiency or inhibition of integrin αvβ6, by administrating the neutralizing antibody, 6.3G9 (10 mg/kg body weight), caused low levels of epithelial cell adhesion molecule and cytokeratin 19 gene messenger RNAs. Also, there were smaller oval cell areas, fewer proliferating ductular epithelial cells, and lower cholestasis serum markers within 2 weeks after DDC treatment. Associated fibrosis was attenuated, as indicated by reduced expression of fibrosis‐related genes, smaller areas of alpha‐smooth muscle actin staining, and low collagen production based on hydroxyproline content and Sirius Red staining. Finally, integrin αvβ6 could bind to CTGF mediating oval cell adhesion to CTGF and fibronection substrata and promoting transforming growth factor (TGF)‐β1 activation in vitro. Conclusions: CTGF and integrin αvβ6 regulate oval cell activation and fibrosis, probably through interacting with their common matrix and signal partners, fibronectin and TGF‐β1. CTGF and integrin αvβ6 are potential therapeutic targets to control DRs and fibrosis in related liver disease. (Hepatology 2015;61:678‐691)

Aptamers against Cells Overexpressing Glypican 3 from Expanded Genetic Systems Combined with Cell Engineering and Laboratory Evolution

Laboratory in vitro evolution (LIVE) might deliver DNA aptamers that bind proteins expressed on the surface of cells. In this work, we used cell engineering to place glypican 3 (GPC3), a possible marker for liver cancer theranostics, on the surface of a liver cell line. Libraries were then built from a six-letter genetic alphabet containing the standard nucleobases and two added nucleobases (2-amino-8H-imidazo[1,2-a][1,3,5]triazin-4-one and 6-amino-5-nitropyridin-2-one), Watson-Crick complements from an artificially expanded genetic information system (AEGIS). With counterselection against non-engineered cells, eight AEGIS-containing aptamers were recovered. Five bound selectively to GPC3-overexpressing cells. This selection-counterselection scheme had acceptable statistics, notwithstanding the possibility that cells engineered to overexpress GPC3 might also express different off-target proteins. This is the first example of such a combination.

A Synthetic Aptamer-Drug Adduct for Targeted Liver Cancer Therapy

AS1411 (previously known as AGRO100) is a 26 nucleotide guanine-rich DNA aptamer which forms a guanine quadruplex structure. AS1411 has shown promising utility as a treatment for cancers in Phase I and Phase II clinical trials without causing major side-effects. AS1411 inhibits tumor cell growth by binding to nucleolin which is aberrantly expressed on the cell membrane of many tumors. In this study, we utilized a simple technique to conjugate a widely-used chemotherapeutic agent, doxorubicin (Dox), to AS1411 to form a synthetic Drug-DNA Adduct (DDA), termed as AS1411-Dox. We demonstrate the utility of AS1411-Dox in the treatment of hepatocellular carcinoma (HCC) by evaluating the targeted delivery of Dox to Huh7 cells in vitro and in a murine xenograft model of HCC.

Linking metabolism and epigenetic regulation in development of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common form of cancer globally and is rarely curable once detected. The 5-year survival rate of patients diagnosed with late-stage HCC may be as low as 27%. HCC is a cancer largely driven by epigenetic changes that arise from exposure to exogenous environmental factors rather than coding sequence mutations. The liver is susceptible to effects from Hepatitis C and Hepatitis B viruses, exposure to aflatoxin and continuous excessive consumption of alcohol. The liver is a highly metabolic organ balancing many vital biochemical processes; exposure to any of the above environmental factors is associated with loss of liver function and is a major risk factor for the development of HCC. Emerging studies aim to examine the underlying metabolic processes that are abrogated in cancer and lead to the altered flux and availability of key metabolites important for epigenetic processes. Metabolites have been shown to act as substrates for many canonical epigenetic regulators. These enzymes are responsible for regulating histone modification, DNA methylation and micro RNA expression. By studying the impact of altered liver metabolism, we may better understand the long-term epigenetic processes, which lead to the development and progression of HCC.

Abstract 2316: GPC3-specific chimeric antigen receptor T cell in combination with Sorafenib as a novel therapeutic treatment for hepatocellular carcinoma

FDA-approved targeted therapy for advanced hepatocellular carcinoma (HCC) is limited with the only option of Sorafenib treatment with severe side effects. Immunotherapy based on adoptive transfer of tumor-specific T cells have demonstrated a promising clinical outcome for solid tumors. In our study, we developed T cells expressing the fourth generation of GPC3-specific chimeric antigen receptor (α-GPC3 CAR) that efficiently eliminated GPC3-positive HCC cells (Huh7 and HepG2) without killing GPC3-negative primary hepatocytes. This CAR was constructed using a lentiviral vector containing CD28, CD27 and 41BB costimulatory domains, a CD3ζ signaling domain fused to a FKBP-iCasp9 apoptosis inducing gene, and a GPC3-specific sequence derived from HN3, a single-domain VH human monoclonal antibody (gift from Mitchell Ho, NCI). After 16h of incubation at the E (α-GPC3 CAR T cells) : T (Huh7 or HepG2 cells) ratio of 1:1, α-GPC3 CAR T cells effectively lysed Huh7 (39%) and HepG2 (77%) but not primary hepatocytes (9%), while the control CAR T cells (α-CD30 CAR) could not initiate a specific lysis on those cells. At the E:T ration of 3:1, the specific killing increased to 78% (Huh7) and 99% (HepG2) while remaining low with primary hepatocytes (11%). Especially at the very low E:T ratio of 1:10, combination with Sorafenib priming (24h, IC10 concentration) prior to CAR T incubation boosted the specific lysis up to 25% (p = 0.0416). These results indicate that combination of GPC3-specificCAR T cells transfer and Sorafenib regimen could be a promising therapeutic option for the treatment of HCC, with the doses of both CAR T and Sorafenib reduced up to 10 times, hence lower side effects while preserving the efficacy of tumor-specific cytotoxicity. Citation Format: Thu Le Trinh, Qunfeng Wu, Lung-Ji Chang, Mitchell Ho, Chen Liu. GPC3-specific chimeric antigen receptor T cell in combination with Sorafenib as a novel therapeutic treatment for hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2316.

Abstract 875: Acquisition of chemoresistance in tumor cells requires crosstalk between dying and remnant live tumor cells via HMGB1

Chemoresistance often develops during drug treatment in cancer patients but its mechanism remains unclear. To understand acquisition of drug resistance, we investigated whether tumor cells dying during drug treatment might signal to neighboring live cells to program themselves for survival in the drug-containing environment. The experimental design was to identify a factor in the supernatants of docetaxel (DTX)-treated human DU145 and PC3 prostate tumor cells that could cause live tumor cells to resist apoptosis and cell death under docetaxel. We discovered that dying cells release HMGB1 which can bind TLR4 or RAGE on live tumor cells causing the induction of cytoplasmic Clusterin (CLU). We demonstrated that CLU is a potent prosurvival protein that can trap Bax from translocating to the mitochondria to cause caspade 3 activation and subsequent apoptosis. Proof of this pathway came from the ability of anti-HMGB1to prevent supernatants from dying cells to induce CLU in newly-plated tumor cells. The receptors for HMGB1 were identified by use of anti-TLR4 and anti-RAGE, which prevented recombinant HMGB1 or dying cell supernatants from inducing CLU in newly-plated tumor cells, causing them to subsequently succumb to DTX toxicity. Moreover, overexpression of anti-sense CLU in tumor cells abrogated their ability to respond to recombinant HMGB1 or dying cell supernatants to induce drug resistance. Most importantly, standard chemotherapeutic agents including gemcitabine, taxol, Ara-C, doxorubicin, cisplatin, etoposide and carboplatin, can cause the release of HMGB1 from dying tumor cells, implicating this crosstalk between dying and live tumor cells as a common pathway towards multi-drug resistance. Citation Format: Junmin Zhou, Xianghong Chen, Danielle L. Gilvary, Melba Marie Tejera, Erika A. Eksioglu, Thu Le Trinh, Sheng Wei, Julie Djeu. Acquisition of chemoresistance in tumor cells requires crosstalk between dying and remnant live tumor cells via HMGB1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 875.

Abstract 418: Epigenetic regulation of glypican-3 in hepatocellular carcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: Glypican 3 (GPC3) is a cell surface heparan sulfate proteoglycan which plays critical roles in the control of cell division and growth regulation. Although repressed in most of adult tissues, GPC3 is found to be upregulated in different types of cancer, including hepatocellular carcinoma (HCC). To address whether the expression of GPC3 in HCC is regulated by epigenetic mechanisms, we studied the DNA methylation profiles of the two CpG islands in the GPC3 promoter by high resolution melting analysis (HRM) and by pyrosequencing. Histone modifications were also examined by ChIP assays to further explore how epigenetic changes contribute to GPC3 expression in HCC. Results: We observed hypomethylation in promoters of 5 different HCC cell lines (which express GPC3) but a partially hypermethylated pattern in HCO2, a HCC cell line in our panel which does not express GPC3. We measured average CpG methylation by pyrosequencing, with the average methylation values for GPC3 expressing cells at 7.74% vs. HCO2 at 43.25% (P<0.01). Similarly, the average methylation frequency in 9 different primary hepatocytes (also no GPC3 expression) is 31.85%, equals to that of HCO2 and much higher than GPC3 expressing cells (P<0.01). In tissues, the methylation profiles of GPC3 promoters are more complicated, where hypo- and hypermethylation could be found in both tumor and non-tumor adjacent tissues. In 34 pairs of tissues, 23 pairs (67.6%) have methylation patterns correlate with expression data. However, treatment of HCO2 with demethylating agent (5-aza-dC) resulted in little reactivation of GPC3 expression, suggesting that DNA methylation may not be the only factor modifying transcription of GPC3. Treatment of HCO2 with histone deacetylase inhibitor trichostatin-A (TSA) resulted in more than 10 folds of GPC3 expression, suggesting histone modification plays a critical role in regulation of GPC3. ChIP analysis in different HCC cell lines as well as primary hepatocytes revealed that the histone marker H3K9Ac (associated with transcription) is present at the GPC3 promoter region of Huh7 but absence in HCO2 and in primary hepatocytes, whereas occupancy of the promoter by repressive markers H3K9Me3 and H3K27Me3 is increased in HCO2 and primary hepatocytes but not Huh7. ChIP assay performed on two pairs of HCC tissues showed similar correlation data between GPC3 expression and histone modification. Conclusions: In this study, we showed that methylation is correlated with transcriptional repression of GPC3 in HCC cell lines, but demethylation of GPC3 promoter is not enough to reactivate the protein expression, suggesting DNA methylation is not the predominant regulatory mechanism for GPC3 gene. Histone modification, on the other hand correlates very tightly with GPC3 expression in HCC cell lines and tumors. Together, our data suggest that epigenetic alterations, including DNA methylation and histone modifications are critical for transcriptional regulation of GPC3 in HCC. Citation Format: Thu Le Trinh, William Puszyk, Chen Liu. Epigenetic regulation of glypican-3 in hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 418. doi:10.1158/1538-7445.AM2014-418

Abstract B97: AS1411-Doxorubicin adduct for targeted liver cancer therapy.

Background: Aptamer-drug conjugates are promising to be applied to targeted cancer therapy, in which anticancer drugs were selectively delivered into cognate cancerous cells, but not to healthy tissues, in order to reduce side effects and enhance therapeutic efficacy. Methodology/Principal Findings: In this study, the technology of drug-DNA adduct (DDA) was utilized to prepare drug-aptamer conjugates. Particularly, an anticancer drug, Doxorubicin (Dox), was conjugated with AS1411, a DNA aptamer which specifically recognizes human hepatocellular carcinoma Huh7 cells. The technology enabled a simple and high-payload conjugation of drugs onto aptamers. The resultant AS1411-Dox adduct maintained selectively recognition ability to target liver cancer cells with strong binding affinity (demonstrated by flow cytometry), was able to selectively deliver the conjugated Dox into target cancer cells (observed with confocal microscopy), and exhibited selective and potent cytotoxicity in target liver cancer cells (evaluated by MTS cell viability assay). More importantly, as the first of its kind, AS1411-Dox adduct demonstrated selectively antitumor therapeutic efficacy of this drug-aptamer conjugation technology in a mouse model. The ability of drug-aptamer adducts to deliver anticancer drugs into target cancer cells, while inhibiting nonspecific drug uptake in nontarget cells, enables this technology promising to be implemented in targeted cancer therapy. Conclusions/Significance: Given the demonstrated ability of targeted liver cancer therapy combined with the aforementioned advantageous features as a simple preparation, the high drug payload capacity, and the wide applicability, this novel drug-DNA adduct technology is anticipated for wide implications for targeted cancer therapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B97. Citation Format: Thu Le Trinh, Guizhi Zhu, Xilin Xiao, Xiaokui Zhang, Kwame Sefah, Weihong Tan, Chen Liu. AS1411-Doxorubicin adduct for targeted liver cancer therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B97.