By Doug Macron
Tekmira Pharmaceuticals this month published preclinical data generated in collaboration with the National Cancer Institute related to the development of a novel siRNA-based treatment of liver cancer.
According to the paper, which appears in Oncogene, the collaborators focused their efforts on CSN5, the fifth subunit of the COP9 signalosome, after an earlier microarray analysis revealed it to be consistently up-regulated in early hepatocellular carcinoma.
CSN5 “is the catalytic center of CSN, an evolutionarily conserved multi-protein complex involved in the control of proteolysis via the ubiquitin proteasome pathway, transcription, protein phosphorylation, and intracellular distribution,” the paper's authors wrote.
“Although the precise molecular mechanisms remain to be elucidated, the most studied function of the CSN is the regulation of protein degradation,” they added. Among the eight CSN subunits, CSN5 plays a “central role” in deneddylation, the stability of key intracellular regulators of survival and apoptosis, and a mediator of transcriptional regulation through mechanisms that have yet to be clearly defined.
“Given the diversity of functions and a large number of effector molecules controlled by CSN5, we hypothesized that the inactivation of the over-expressed CSN5 gene could result in re-establishment of numerous anti-oncogenic programs involved in the control of cell proliferation and apoptosis, and whereby be effective as an anti-cancer strategy,” the Tekmira and NCI investigators noted in Oncogene.
To test this, the researchers designed three CSN5-specific siRNAs and transfected them into HCC cell lines. One siRNA in particular down-regulated its target, an effect that was correlated with a “remarkable” decrease in target protein, while suppressing HCC cell growth more than the other two, the paper states.
Further in vitro testing revealed that the siRNA's ability to suppress the level of CSN5 was “sufficient to interfere with CSN function, reflected by “the accumulation of neddylated Cullin 1 and changes in the protein levels of CSN-controlled substrates SKP2, p53, p27, and nuclear factor-jB,” according to the paper.
Meanwhile, transcriptomic analysis showed that the anti-proliferative effect of CSN5 knockdown was “driven by a common subset of molecular alterations including down-regulation of cyclin-dependent kinase 6 and integrin b1, which were functionally interconnected with key oncogenic regulators MYC and TGFb1 involved in the control of proliferation, apoptotic cell death, and HCC progression.”
Western blots were in line with the microarray findings, revealing that CSN5 depletion boosted phosphorylation of Smad 2 and 3, which mediate TGFb1 signaling; decreased the protein levels of ITGB1, CDK6, and cyclin D1; and reduced the expression of anti-apoptotic Bcl-2, while elevating the levels of pro-apoptotic Bak,” the authors wrote.
To extend its findings in vivo, the team chemically modified the CSN5-targeting siRNA and encapsulated it in a Tekmira-developed lipid nanoparticle. The researchers then administered the formulated siRNAs to orthotopic mouse models of HCC.
“The results showed that in a control group treated with [a control siRNA], tumors grew very rapidly and at the end point of the study occupied a significant portion of liver parenchyma,” according to the paper. “In addition, the majority of mice developed ascites, reflecting impairment of liver function characteristic for the end stage of liver cancer disease.”
In contrast, the siRNA molecule targeting CSN5 “effectively inhibited the hepatic tumor growth ... and significantly improved well-being of [the] animals,” the study found. “Consistent with this, mice receiving siRNA therapy showed a better histology with single and much smaller tumors, as well as reduction in liver-to-body ratios as [a] reflection of the reduced tumor burden.”
The paper's authors noted that among their most important findings was that combining Tekmira's lipid nanoparticle technology with the 2'-O-methyl modifications made to the siRNA effectively protected the drug's RNAi payload from glomerular filtration and serum nucleases, and yielded “potent silencing of target mRNA without cytokine induction, toxicity, or off-target effects.”
Using Tekmira's delivery technology also further enabled the researchers to reduce the dosing regimen by up to 3 mg/kg — 10-fold less than required by a standard siRNA dose administered without a carrier.
“Although more work is needed to optimize the therapeutic dose of [the siRNA drug] for different stages of HCC disease, our work demonstrates the potency of systemic RNA interference targeting of CSN5 without an overt toxicity in a mouse model of orthotopic transplantation of human HCC cells and suggests its clinical utility for treatment of HCC disease,” the team concluded.
"We look forward to continuing our collaborative work with the NCI to identify novel cancer targets and demonstrate anti-tumor activity by silencing these genes through RNA interference with the goal of advancing new oncology product candidates," Tekmira President and CEO Mark Murray added in a statement.
The Oncogene paper comes about five months after the company hinted that it might test TKM-PLK1 as a liver cancer drug with the NCI. TKM-PLK1 is already in phase I testing for solid tumors outside the liver.
Last year, Murray said that the company was exploring the idea of running a liver cancer-specific clinical trial of TKM-PLK1 in collaboration with the NCI's surgical branch (GSN 11/18/2010).
He said doing so would give Tekmira access to a population of liver-cancer patients “where tumor biopsies allowing measurement of RNAi drug activity may be more readily available."
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