The most recent reeasearch includes (1)Cholangiocarcinoma is a heterogeneous disease with a poor outcome that accounts for 5%-10% of primary liver cancers. We characterized its genomic and genetic features and associated these with patient responses to therapy. We profiled the transcriptomes from 104 surgically resected cholangiocarcinoma samples collected from patients in Australia, Europe, and the United States;epithelial and stromal compartments from 23 tumors were laser capture microdissected. We analyzed mutations in KRAS, epidermal growth factor receptor (EGFR), and BRAF in samples from 69 tumors. Changes in gene expression were validated by immunoblotting and immunohistochemistry;integrative genomics combined data from the patients with data from 7 human cholangiocarcinoma cell lines, which were then exposed to trastuzumab and lapatinib. Patients were classified into 2 subclasses, based on 5-year survival rate (72% vs 30%;P .0007), time to recurrence (13.7 vs 22.7 months;P .001), and the absence or presence of KRAS mutations (24.6%), respectively. Class comparison identified 4 survival subgroups (SGI-IV;P .03);SGIII was characterized by genes associated with proteasomal activity and the worst prognosis. The tumor epithelium was defined by deregulation of the HER2 network and frequent overexpression of EGFR, the hepatocyte growth factor receptor (MET), pRPS6, and Ki67, whereas stroma was enriched in inflammatory cytokines. Lapatinib, an inhibitor of HER2 and EGFR, was more effective in inhibiting growth of cholangiocarcinoma cell lines than trastuzumab. In conclusion, we provide insight into the pathogenesis of cholangiocarcinoma and identify previously unrecognized subclasses of patients, based on KRAS mutations and increased levels of EGFR and HER2 signaling, who might benefit from dual-target tyrosine kinase inhibitors. In conclusion, we provide insight into the pathogenesis of cholangiocarcinoma and identify previously unrecognized subclasses of patients, based on KRAS mutations and increased levels of EGFR and HER2 signaling, who might benefit from dual-target tyrosine kinase inhibitors. The group of patients with the worst prognosis was characterized by transcriptional enrichment of genes that regulate proteasome activity, indicating new therapeutic targets.(2) We have previously demonstrated therapeutic effects of lipid nanoparticles (LNP) loaded with single siRNA targeting CSN5 or WEE1 against human HCC cell lines in an orthotropic mouse models. To test the safety and the efficacy of a combinatorial versus single siRNA therapy in the orthotopic mouse model and to identify molecular mechanism(s) involved in therapeutic responses by global transcriptome analyses. LNP formulations of chemically modified siRNAs targeting CSN5 and WEE1 were produced by Tekmira Pharmaceuticals. Safety was assessed in ICR mice after 9 injections. SCID-beige mice were used for intra-hepatic (Huh7-luciferase) tumor transplantation. Mice with established tumors were treated intravenously with 2 mg/kg of a single siRNA + 2 mg/kg betagal siRNA or 2 mg/kg each of siCSN5:siWEE1 siRNA coencapsulated in the same LNP. Tumors were assayed following 1 to 9 injected doses. Tumor progression in the Huh7 orthotopic model was monitored by bioluminescence imaging and metastases were evaluated at endpoint. Safety data show that combinatorial siRNA is well tolerated compared to single or control siRNA. We observed significant inhibition of tumor growth and metastases in mice treated with active siRNAs compared to LNP containing a non-targeting control siRNA. Significant targeted silencing was observed in tumors after single or repeat administration with no interference between the siRNAs for the CSN5:WEE1 combination. Potency was not lost with siCSN5:siWEE1 LNP, relative to the most efficacious single agent. Microarray analyses of the tumors demonstrate an extensive difference of gene expression between treatment groups. Interestingly,the microarray analyses of the surrounding liver show a minimal modification of gene expression in this non-tumor tissue. In conclusion we demonstrate that LNP-based combinatorial siRNA therapy is safe and effective in a human mouse model of HCC, with a significant decrease of tumor size associated with a massive downregulation of the targeted genes. Global gene expression of the surrounding liver is minimally affected by this therapy compared with what seen in the tumor.(3)We have previously shown that histone deacetylase 2 (HDAC2), which regulates gene expression by deacetylating histones, is upregulated in human HCC. In this study we have investigated if small interfering (si) RNA targeting of HDAC2 could affect the course of HCC progression with an enhanced target-specificity. Our results show that siRNA silencing of HDAC2 caused a strong induction of apoptosis and inhibition of cell growth in HCC cell lines. Comparison of isoform selectivity between HDAC2 siRNA and the HDAC inhibitor SAHA treatment revealed that HDAC2 siRNA specifically downregulates target protein HDAC2 only. However SAHA treatment did not demonstration any significant isoform-selectivity, increasing the level of acetylated histones and consequently caused changes in the expression of large number of genes. siRNA induced depletion of HDAC2 revealed that the anti-proliferative effect was caused by gene set that including up regulation of p27 and down regulation of CDK6 and BCL-2. Systemic delivery of a modified HDAC2 siRNA by lipid nanoparticles (LNP) significantly suppressed growth of human HCC cells in mice liver. These findings suggest that LNP based selective siRNA targeting of HDAC2 can be a promising substitute for small molecular inhibitors of HDAC2 in systemic therapy of HCC.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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