We validated the therapeutic potential of 8 genes (COP1, CSN5, HDAC2, R1 (RAM2), RBX1, WEE1, FOXM1 and CDK4), all with high hazard ratio for survival. For each gene, three specific siRNAs encapsulated in stable-nucleic-acid-lipid-particle (SNALP) were designed and tested for growth inhibition in two HCC cell lines (Huh7 and HepG2) as determined by MTT and TUNEL assays. Reduction in target RNA was confirmed by qRT-PCR analysis. The siRNA targeting of COP1, CSN5 and HDAC2 genes in the HCC cells restored the levels of p53 and its target p21, suggesting that siRNA-induced apoptosis was p53-dependent. siRNAs showing therapeutic activity in cell culture were further evaluated in vivo using a luciferase-based reporter cell line Huh7luc+ and an orthotopic xenograft model. Chemically modified siRNAs were systemically delivered to liver through tail vein injections. SNALP was selected as an optimal carrier for siRNA delivery into the mouse because of the enhanced stability, lack of toxicity and smaller effective dose. Further, SNALP technology for in vivo siRNA delivery may be an effective way to treat human HCC. Future plans include (i) generating more HCC cell lines expressing luciferase for in vivo validation of the therapeutic activity of selected genes;and (ii) understanding the molecular mechanisms underlying growth inhibition and apoptotic induction in Huh7 and HepG2 cells triggered by target gene silencing, using a microarray approach. The ultimate goal of this project is to use siRNA-SNALP therapeutic agents for the treatment of HCC patients. As a first step, Investigative New Drug (IND) applications will be filed for the two most effective siRNAs (COP1 and WEE1) in collaboration with Tekmira Pharmaceuticals and Dr. Avital, Surgery Branch, NCI. Currently progress includes: (1)We have previously demonstrated that constitutively photomorphogenic 1 (COP1), which regulates p53 activity by ubiquitination, is frequently overexpressed in human HCC. Here we examined whether molecular targeting of COP1 by small interfering (si) RNA can affect the course of HCC progression. COP1-1 was selected as the most effective target siRNAs in terms of growth inhibition and apoptotic induction in several HCC cell lines. The growth inhibition occurred both in HCC cells that retain wild-type p53 or express mutant p53 (Y220C or R249S) whereas p53-null Hep3B cells were resistant. Analysis of COP1 knockdown gene expression signatures by microarray revealed that the anti-proliferative effect was driven by a common subset of molecular alterations including p53-associated functional network. Systemic delivery of a modified COP1siRNA by SNALPs significantly suppressed neoplastic growth in liver, without unwanted immune response in an orthotopic xenograft model. These findings provide the first proof of principle that COP1 is a promising target for systemic therapy of HCC;(2) We have recently identified an elevated expression of histone deacetylase 2 (HDAC2) in primary human hepatocellular carcinoma (HCC) and HCC derived cell lines. We have tested if the downregulation of HDAC2 by siRNA can affect the course of HCC progression in vitro and in vivo. To inactivate HDAC2 gene expression, three HDAC2-specific siRNAs (HDAC2-1, HDAC2-2 and HDAC2-3) were designed and tested for growth inhibition in Huh7 and HepG2 HCC cell lines as determined by MTT assay, FACS analysis and microscopy. To obtain insights into molecular changes caused by HDAC2 silencing, global changes in gene expression were examined by illumina microarray. For in vivo evaluation of HDAC2 as a therapeutic target, we employed orthotopic xenograft model using luciferase-expressing HCC reporter cell line Huh7-luc+ and SNALP as an optimal carrier of siRNA into liver. The HDAC2-1siRNA was the most effective in inhibiting Huh7 and HepG2 cell growth (68% and 71%, respectively) which was paralleled by a similar decrease in the levels of target mRNA and protein. HDAC2-deficient cells also exhibited a 1.9-fold increase in apoptosis through activation of caspase-3. The comparison of gene expression profiles in HepG2 cells treated with either control siRNA or HDAC2-1siRNA identified 299 differentially expressed genes. Consistent with in vitro observations, genes functionally involved in apoptosis, such as CDKN1A, SOCS2, TP53I3, and BTG2, were up-regulated while genes associated with cellular metabolic process (e.g. HOXD1, PAK2, SMAD9, CDK4, PCK2 and SKP2) were down-regulated. HDAC2 3/7siRNA, a chemically modified variant of HDAC2-1siRNA displaying a minimal induction of IL-6 in murine Flt3L dendricite cultures, was selected for in vivo treatment of Huh7-luc+-derived HCC. Administration of SNALP- HDAC2 3/7siRNA effectively suppressed xenograft growth as compared to the SNALP-control siRNA treatment. Taken together, these results indicate that HDAC2 is an important regulator of HCC cell growth and survival, and may be an attractive target for systemic therapy of HCC;and (3) we have performed a comprehensive characterization of epigenomic modulation caused by zebularine, an effective DNA methylation inhibitor, in primary human liver cancer and liver cancer cell lines. Using transcriptomic and epigenomic profiling, we identified a zebularine signature that classified liver cancer cell lines into two major subtypes with different drug-responses. In drug-sensitive cell lines, zebularine caused inhibition of proliferation coupled with increased apoptosis, whereas drug-resistant cell lines were associated with upregulation of oncogenic networks (e.g. E2F1, MYC, and TNF) driving liver cancer growth in vitro and in mice. Assessment of zebularine-based therapy in xenograft mouse models demonstrated potent therapeutic effects against tumors established from zebularine-sensitive but not zebularine-resistant liver cancer cells leading to increased survival and decreased pulmonary metastasis. Integration of zebularine gene expression and demethylation response signatures differentiated patients with HCC according to their survival and disease recurrence and identified a subclass of patients within the poor survivors likely to benefit from therapeutic agents that target the cancer epigenome.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC011175-02
Application #
8157673
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
2010
Total Cost
$534,598
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
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