Dharmacon siGENOME® SMARTpool® siRNA Libraries Used in Screen to Identify Chemosensitizer Genes for Cancer Drugs

Allison Sterling, Ph.D.

In the ten years since RNA interference (RNAi) was discovered by Fire and Mello, an enormous field of research has emerged revolving around this endogenous pathway.¹ RNAi research initially focused on exploring the RNAi mechanism but rapidly expanded to optimizing synthetic versions of the main component in the process, short interfering RNA molecules (siRNA) for use in functional genomic studies and therapeutic applications. Synthetic siRNA is already in FDA trials as a therapeutic for the treatment of macular degeneration and respiratory syncytial virus.² Many other diseases are also being investigated, including diabetic retinopathy, viral lung infections, Parkinson’s, in vivo applications in oncology and liver diseases, AIDS, hepatitis C, Huntington’s, hair removal, etc.^3,4

While the idea to use RNAi for direct therapeutic application is well established, the indirect therapeutic application of siRNA is emerging as chemosensitizers to cancer drugs. Chemosensitizers are drugs which make tumor cells more sensitive to the effects of chemotherapy. The necessity for chemosensitizers is driven by the increasing number of patients with drug resistance tumors, either innate or acquired.⁵ One chemotherapeutic drug, paclitaxel (developed commercially as Taxol® by Bristol-Myers Squibb) has shown widespread resistance and incomplete responses in patients. Paclitaxel functions by disrupting mitosis in cancer cells to effectively prevent tumor cell growth or proliferation. Thus much recent research has focused on understanding the cause behind paclitaxel resistance. Recently, two independent studies were published using Dharmacon® siGENOME® SMARTpool® siRNA Libraries to investigate both the mechanism of paclitaxel resistance as well as the utility of siRNA as paclitaxel chemosensitizers.^5,6

A study by Whitehurst et al. used a genome-wide Dharmacon® siGENOME® SMARTpool® siRNA library screen to identify genes that sensitize cancer cells (specifically non-small-cell lung cancer) to paclitaxel.⁶ The initial siRNA screen identified a set of 87 candidate genes that sensitize cells to paclitaxel. The 87 candidate genes were grouped into like functional groups (e.g. proteasome, Ras family, etc.) and follow-up studies conducted on six targets representative of the functional groups. Using Dharmacon siGENOME SMARTpool siRNA reagents targeting the six genes, the extent of paclitaxel chemosensitization by target gene depletion was investigated. A broad range of concentrations of paclitaxel as well as two other chemotherapeutic agents were examined and it was found that the siRNA-inhibited cells were only sensitized to low levels paclitaxel, a drug that impairs mitotic spindle assembly. The low levels of paclitaxel were actually concentrations 1,000-fold lower than normally required for significant response. The authors went on to further investigate the sensitization mechanism and its synergy with paclitaxel’s mode of action and confirmed the direct role of mitotic genes in both pathways.

In a similar, but more focused investigation, Swanton et al. examined both the mechanism of paclitaxel resistance as well as genes that sensitize cancer cells using Dharmacon siGENOME SMARTpool siRNA Protein Kinase and custom Ceramide Kinase Libraries.⁵ Forty-three siRNAs that antagonize paclitaxel activity were identified in the initial Kinome siRNA screen, with a majority of the genes relating to mitotic function. Twenty-six genes were reassayed across three cell lines and validated as paclitaxel antagonizers. As paclitaxel disrupts mitosis in cancer cells to effectively prevent tumor cell growth or proliferation, paclitaxel antagonists reduce the level of mitotic arrest (mitotic index) and consequently the efficacy of paclitaxel. Thus, it was reasoned that paclitaxel antagonists might cause defects in the mitotic process such as spindle checkpoint defects and induce polyploidy in the absence of paclitaxel. It was found that 17/20 of the validated siRNAs in HCT-116 cells demonstrated an increase in polyploidy without drug treatment. Further studies found that the validated siRNAs also induced additional chromosomal instabilities (CIN), such as multinucleation and abnormalities in centromere size and number. These results suggest a possible relationship between CIN in cancer cells and paclitaxel resistance. From the initial kinome siRNA screen, the paclitaxel sensitivity index was calculated and ceramide transport protein (COL4A3BP) identified as a paclitaxel chemosensitizer. A second screen using the Ceramidome siRNA library was conducted and confirmed the role of ceramide proteins as paclitaxel antagonists. Further experiments found that COL4A3BP expression was increased in drug-resistant cell lines and residual tumor (following paclitaxel treatment) suggesting that it could be a target for chemotherapy-resistant cancers.

In summary, these two studies have shown that synthetic siRNA has a burgeoning role not only as a tool for drug mechanism exploration, but also as indirect therapeutics such as chemosensitizers. Additionally, synthetic siRNA genome-wide libraries facilitate functional analysis at unprecedented speeds – from understanding mechanisms to developing therapeutics in very short times.

References

1. Fire, A., Xu, S., et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature (1998). 391(6669):806-11.
2. Sah D. Therapeutic potential of RNA interference for neurological disorders. Life Sciences (2006). 79(19): 1773–80.
3. Behlke, M.A. Progress Towards in Vivo Use of siRNAs. Molecular Therapy (2006). 13(4): 644-70.
4. Uprichard, S.L. The therapeutic potential of RNA interference. FEBS Letters (2005). 579 :5996–6007.
5. Swanton C, Marani M, et al. Regulators of mitotic arrest and ceramide metabolism are determinants of sensitivity to paclitaxel and other chemotherapeutic drugs. Cancer Cell (2007). 11(6):498-512.
6. Whitehurst AW, Bodemann BO, et al. Synthetic lethal screen identification of chemosensitizer loci in cancer cells. Nature (2007). 46(7137):815-9.