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Molecular targeting of CSN5 in human hepatocellular carcinoma: a mechanism of therapeutic response.

Lee YH, Judge AD, Seo D, Kitade M, Gómez-Quiroz LE, Ishikawa T, Andersen JB, Kim BK, Marquardt JU, Raggi C, Avital I, Conner EA, MacLachlan I, Factor VM, Thorgeirsson SS - Oncogene (2011)

Bottom Line: Consistent with microarray analysis, western blotting revealed that CSN5 depletion increased phosphorylation of Smad 2/3, key mediators of TGFβ1 signaling, decreased the protein levels of ITGB1, CDK6 and cyclin D1 and caused reduced expression of anti-apoptotic Bcl-2, while elevating the levels of pro-apoptotic Bak.A chemically modified variant of CSN5 siRNA was then selected for in vivo application based on the growth inhibitory effect and minimal induction of unwanted immune response.Taken together, these results indicate that CSN5 has a pivotal role in HCC pathogenesis and maybe an attractive molecular target for systemic HCC therapy.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Experimental Carcinogenesis, Center for Cancer Research, National Cancer Institute, National Institute of Health, Bethesda, MD, USA.

ABSTRACT
Development of targeted therapy for hepatocellular carcinoma (HCC) remains a major challenge. We have recently identified an elevated expression of the fifth subunit of COP9 signalosome (CSN5) in early HCC as compared with dysplastic stage. In the present study, we explored the possibility of CSN5 being a potential therapeutic target for HCC. Our results show that CSN5 knockdown by small-interfering (si) RNA caused a strong induction of apoptosis and inhibition of cell-cycle progression in HCC cells in vitro. The down-regulation of CSN5 was sufficient to interfere with CSN function as evidenced by the accumulation of neddylated Cullin 1 and changes in the protein levels of CSN-controlled substrates SKP2, p53, p27 and nuclear factor-κB, albeit to a different degree depending on the HCC cell line, which could account for the CSN5 knockdown phenotype. The transcriptomic analysis of CSN5 knockdown signature showed that the anti-proliferative effect was driven by a common subset of molecular alterations including down-regulation of cyclin-dependent kinase 6 (CDK6) and integrin β1 (ITGB1), which were functionally interconnected with key oncogenic regulators MYC and TGFβ1 involved in the control of proliferation, apoptotic cell death and HCC progression. Consistent with microarray analysis, western blotting revealed that CSN5 depletion increased phosphorylation of Smad 2/3, key mediators of TGFβ1 signaling, decreased the protein levels of ITGB1, CDK6 and cyclin D1 and caused reduced expression of anti-apoptotic Bcl-2, while elevating the levels of pro-apoptotic Bak. A chemically modified variant of CSN5 siRNA was then selected for in vivo application based on the growth inhibitory effect and minimal induction of unwanted immune response. Systemic delivery of the CSN5 3/8 variant by stable-nucleic-acid-lipid particles significantly suppressed the tumor growth in Huh7-luc+ orthotopic xenograft model. Taken together, these results indicate that CSN5 has a pivotal role in HCC pathogenesis and maybe an attractive molecular target for systemic HCC therapy.

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Selection of CSN5 3/8 for in vivo application based on the inhibition of tumor cell growth and minimal cytokine induction. (a) Inhibition of Huh7-luc+ cell growth after transfection with 15 nM of SNALP-formulated CSN5-2 (native) or its modified variants (CSN5-3/6~9, CSN5-4/6~9, CSN5-5/6~9). The siRNA transfectants were examined by MTT assay at 4 d after treatment. **, P < 0.01 (n=3) by Bootstrap t-test. SNALP-Luc, SNALP-fromulated siRNA targeting luciferase. (b) Quantification of IL-6 level after CSN5 targeting. Culture supernatants of Flt3L-derived dendrocytes were assayed for IL-6 using ELISA at 24 h after siRNA treatment. The data are shown as the means ± S.D. of triplicate experiments.
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Figure 5: Selection of CSN5 3/8 for in vivo application based on the inhibition of tumor cell growth and minimal cytokine induction. (a) Inhibition of Huh7-luc+ cell growth after transfection with 15 nM of SNALP-formulated CSN5-2 (native) or its modified variants (CSN5-3/6~9, CSN5-4/6~9, CSN5-5/6~9). The siRNA transfectants were examined by MTT assay at 4 d after treatment. **, P < 0.01 (n=3) by Bootstrap t-test. SNALP-Luc, SNALP-fromulated siRNA targeting luciferase. (b) Quantification of IL-6 level after CSN5 targeting. Culture supernatants of Flt3L-derived dendrocytes were assayed for IL-6 using ELISA at 24 h after siRNA treatment. The data are shown as the means ± S.D. of triplicate experiments.

Mentions: The next step was to validate if systemic silencing of CSN5 could suppress liver tumor growth. To prevent immune activation and enhance siRNA stability in vivo, native CSN5-2siRNA was chemically modified by selective incorporation of 2′-O-methyl (2′OMe) uridine or guanosine nucleosides into one strand of the siRNA duplex, and encapsulated into SNALP. A modified siRNA was then screened for in vivo application in terms of Huh7-luc+ cell growth and cytokine induction using murine Flt3L dendricytes isolated from mouse bone marrow. Among the variants tested, CSN5 3/8 sequence was the most effective in inhibiting tumor cell growth (about 80%) (Figure 5a). In addition, the modified sequence caused a minimal induction of IL-6 as compared to the treatment with native CSN5-2siRNA while injection of empty SNALP had little effect on IL-6 levels (Figure 5b).


Molecular targeting of CSN5 in human hepatocellular carcinoma: a mechanism of therapeutic response.

Lee YH, Judge AD, Seo D, Kitade M, Gómez-Quiroz LE, Ishikawa T, Andersen JB, Kim BK, Marquardt JU, Raggi C, Avital I, Conner EA, MacLachlan I, Factor VM, Thorgeirsson SS - Oncogene (2011)

Selection of CSN5 3/8 for in vivo application based on the inhibition of tumor cell growth and minimal cytokine induction. (a) Inhibition of Huh7-luc+ cell growth after transfection with 15 nM of SNALP-formulated CSN5-2 (native) or its modified variants (CSN5-3/6~9, CSN5-4/6~9, CSN5-5/6~9). The siRNA transfectants were examined by MTT assay at 4 d after treatment. **, P < 0.01 (n=3) by Bootstrap t-test. SNALP-Luc, SNALP-fromulated siRNA targeting luciferase. (b) Quantification of IL-6 level after CSN5 targeting. Culture supernatants of Flt3L-derived dendrocytes were assayed for IL-6 using ELISA at 24 h after siRNA treatment. The data are shown as the means ± S.D. of triplicate experiments.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3140552&req=5

Figure 5: Selection of CSN5 3/8 for in vivo application based on the inhibition of tumor cell growth and minimal cytokine induction. (a) Inhibition of Huh7-luc+ cell growth after transfection with 15 nM of SNALP-formulated CSN5-2 (native) or its modified variants (CSN5-3/6~9, CSN5-4/6~9, CSN5-5/6~9). The siRNA transfectants were examined by MTT assay at 4 d after treatment. **, P < 0.01 (n=3) by Bootstrap t-test. SNALP-Luc, SNALP-fromulated siRNA targeting luciferase. (b) Quantification of IL-6 level after CSN5 targeting. Culture supernatants of Flt3L-derived dendrocytes were assayed for IL-6 using ELISA at 24 h after siRNA treatment. The data are shown as the means ± S.D. of triplicate experiments.
Mentions: The next step was to validate if systemic silencing of CSN5 could suppress liver tumor growth. To prevent immune activation and enhance siRNA stability in vivo, native CSN5-2siRNA was chemically modified by selective incorporation of 2′-O-methyl (2′OMe) uridine or guanosine nucleosides into one strand of the siRNA duplex, and encapsulated into SNALP. A modified siRNA was then screened for in vivo application in terms of Huh7-luc+ cell growth and cytokine induction using murine Flt3L dendricytes isolated from mouse bone marrow. Among the variants tested, CSN5 3/8 sequence was the most effective in inhibiting tumor cell growth (about 80%) (Figure 5a). In addition, the modified sequence caused a minimal induction of IL-6 as compared to the treatment with native CSN5-2siRNA while injection of empty SNALP had little effect on IL-6 levels (Figure 5b).

Bottom Line: Consistent with microarray analysis, western blotting revealed that CSN5 depletion increased phosphorylation of Smad 2/3, key mediators of TGFβ1 signaling, decreased the protein levels of ITGB1, CDK6 and cyclin D1 and caused reduced expression of anti-apoptotic Bcl-2, while elevating the levels of pro-apoptotic Bak.A chemically modified variant of CSN5 siRNA was then selected for in vivo application based on the growth inhibitory effect and minimal induction of unwanted immune response.Taken together, these results indicate that CSN5 has a pivotal role in HCC pathogenesis and maybe an attractive molecular target for systemic HCC therapy.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Experimental Carcinogenesis, Center for Cancer Research, National Cancer Institute, National Institute of Health, Bethesda, MD, USA.

ABSTRACT
Development of targeted therapy for hepatocellular carcinoma (HCC) remains a major challenge. We have recently identified an elevated expression of the fifth subunit of COP9 signalosome (CSN5) in early HCC as compared with dysplastic stage. In the present study, we explored the possibility of CSN5 being a potential therapeutic target for HCC. Our results show that CSN5 knockdown by small-interfering (si) RNA caused a strong induction of apoptosis and inhibition of cell-cycle progression in HCC cells in vitro. The down-regulation of CSN5 was sufficient to interfere with CSN function as evidenced by the accumulation of neddylated Cullin 1 and changes in the protein levels of CSN-controlled substrates SKP2, p53, p27 and nuclear factor-κB, albeit to a different degree depending on the HCC cell line, which could account for the CSN5 knockdown phenotype. The transcriptomic analysis of CSN5 knockdown signature showed that the anti-proliferative effect was driven by a common subset of molecular alterations including down-regulation of cyclin-dependent kinase 6 (CDK6) and integrin β1 (ITGB1), which were functionally interconnected with key oncogenic regulators MYC and TGFβ1 involved in the control of proliferation, apoptotic cell death and HCC progression. Consistent with microarray analysis, western blotting revealed that CSN5 depletion increased phosphorylation of Smad 2/3, key mediators of TGFβ1 signaling, decreased the protein levels of ITGB1, CDK6 and cyclin D1 and caused reduced expression of anti-apoptotic Bcl-2, while elevating the levels of pro-apoptotic Bak. A chemically modified variant of CSN5 siRNA was then selected for in vivo application based on the growth inhibitory effect and minimal induction of unwanted immune response. Systemic delivery of the CSN5 3/8 variant by stable-nucleic-acid-lipid particles significantly suppressed the tumor growth in Huh7-luc+ orthotopic xenograft model. Taken together, these results indicate that CSN5 has a pivotal role in HCC pathogenesis and maybe an attractive molecular target for systemic HCC therapy.

Show MeSH
Related in: MedlinePlus