Ankita Brahmaroutu- FGF8b

RNA Aptamer Selection against FGF8b: diagnostic tool for neuronal differentiation

Fall 2012

Abstract:

It is estimated that over one million people will be diagnosed with cancer and over half a million men and women will die of cancer of all sites this year. Particularly, it is estimated that over 13,000 people will die of brain and nervous system cancers this year (Howlader et al. 2012). Fibroblast growth factor 8b (FGF8b) is an oncogene responsible in the formation of tumors. In the specific case of neuronal differentiation, FGF8b signaling is largely involved in posterior neural formation when studied in Xenopus. It is found that “FGF8 is necessary for proper gastrulation and formation of mesoderm” as well (Fletcher et al. 2006).

It has also been observed that the loss of FGF8b reduces areas of the hindbrain and spinal cord during development (Fletcher et al. 2006). Fgf8b activates the “Ras-extracellular signal-regulated kinase (ERK) pathway,” which induces cerebellar development (Sato et al. 2004). Besides neuronal sites, overexpression of FGF8b was found in the biopsies of esophagus and prostate cancers (Lui et al. 2010, Zhong et al. 2011). “Upregulation of the FGF8b signaling system seen in many cancers may contribute to the proliferation of the malignant cells in vivo” (Viklund et al. 2006). Therefore, an overexpression of FGF8b can lead to uncontrolled cell growth, leading to tumors. An aptamer to be used as a diagnostic tool to detect the action of neuronal differentiation could be used to quickly identify where and when overexpression of this oncogene begins.

Aptamers are single-stranded RNA or DNA oligonucleic acid molecules which are selected for different targets. Aptamer uses have expanded greatly since the discovery of aptamers in 1990 (Tuerk and Gold 1990, Ellington and Szostak 1990). The applications of aptamers include therapeutics, systems biology, diagnostics, target validation, and drug delivery (Proske et al. 2005).  Due to their three-dimension folded shape, aptamers offer high selectivity and sensitivity. They have a high affinity for a larger range of molecules: toxins, prions, and ion targets (Tombelli et al. 2004). Advantages of aptamers over monoclonal antibodies are that they can differentiate between closely related targets, include chemically produced oligonucleotide libraries, and have durable structure (Stoltenburg et al. 2007). The selection of this aptamer was applied through the SELEX (systematic evolution of ligands by exponential enrichment) process, which, starting from large libraries of oligonucleotides, allows the isolation of the high affinity nucleic acids by the process of in vitro selection and amplification through polymerase chain reaction (Proske et al. 2005).

Specific Aim 1: Selection of RNA aptamers against FGF8b

FGF8b is an oncogene responsible in the formation of tumors. When FGF8b is overexpressed, tumors are formed. Once a particular sample of either early stage to late stage cancer cells has been taken in vitro, an aptamer for FGF8b could be introduced inside the cells. Where there is a proliferation of aptamer binding (tagged with a fluorescent), a cancerous cell may be present. This will enable surgeons to be able to remove cells in sites where these samples were taken from (with continuous FGF8b aptamer binding) and prevent the spread of cancerous cells. Figure 1 is a pictorial representation of how cells with aptamer binding may be identified.

Figure 1:

The figure above is a very simplified idea of how an aptamer for FGF8b will help to identify cancerous neuron cells. Tagged with a fluorescent, the aptamers will bind to the FGF8b oncogene. A large amount of aptamer binding will indicate an overexpression of the oncogene, and thus the cells can be isolated and therapy can start for the patient.

Legend:

Green	Aptamers binding to FGF8b in a cell
Beige	Neuron cell

Product: FGF8b Human Recombinant

Amount: 25 μg

Website: http://www.prospecbio.com/FGF_8_Human_2_15/?gclid=CMr2h5mz-bECFSaHtgodO28AtQ

Catalog Number: CYT-714

Price: $130

References:

1.       Stoltenburg R, Reinemann C, Strehlitz B. SELEX--a (r)evolutionary method to generate high-affinity nucleic acid ligands, Biomolecular Engineering 2007; 24 (4): 381-403.

2.       Ellington A, Szostak J. In vitro selection of RNA molecules that bind specific ligands, Nature 1990; 346 (6287): 818-822.

3.       Tombelli S, Minunni M, Mascini M. Analytical applications of aptamers, Biosens Bioelectron 2004; 20 (12): 2424-2434. Available from: http://www.ncbi.nlm.nih.gov/pubmed/15854817. Accessed  2012 September 3.

4.       Proske D, Blank M, Buhmann R, Resch A. Aptamers—basic research, drug development, and clinical applications, Applied Microbiology and Biotechnology 2005; 69 (4): 367-374. Available from: http://www.springerlink.com/content/p77317t5u801gj31/. Accessed  2012 September 1.

5.       Lui VW, Yau DM, Cheung CS, Wong SC, Chan AK, Zhou Q, Wong EY, Lau CP, Lam EK, Hui EP, Hong B, Hui CW, Chan AS, Ng PK, Ng YK, Lo KW, Tsang CM, Tsui SK, Tsao SW, Chan AT. FGF8b oncogene mediates proliferation and invasion of Epstein-Barr virus-associated nasopharyngeal carcinoma cells: implication for viral-mediated FGF8b upregulation, Oncogene 2011 Mar 31;30(13):1518-30.

6.       Fletcher R, Baker J, Harland R. FGF8 spliceforms mediate early mesoderm and posterior

neural tissue formation in Xenopus, Development 133, 1703-1714 (2006).

7.       Sato T, Joyner AL, Nakamura H. How does Fgf signaling from the isthmic organizer induce midbrain and cerebellum development?, Dev Growth Differ. 2004 Dec;46(6):487-94.

8.       Howlader N, Noone AM, Krapcho M, Neyman N, Aminou R, Altekruse SF, Kosary CL, Ruhl J, Tatalovich Z, Cho H, Mariotto A, Eisner MP, Lewis DR, Chen HS, Feuer EJ, Cronin KA (eds). SEER Cancer Statistics Review, 1975-2009 (Vintage 2009 Populations), National Cancer Institute.

9.       Zhong C, Saribekyan G, Liao C, Cohen M, Roy-Burman P. Cooperation between FGF8b Overexpression and PTEN Deficiency in Prostate Tumorigenesis. Cancer Res February 15, 2006 66; 2188.

10.   Viklund L, Vorontsova N, Henttinen T, Salmivirta M. Syndecan-1 regulates FGF8b responses in S115 mammary carcinoma cells. Growth Factors. 2006 Jun;24(2):151-7.

Proposal
Progress Report 1

Progress Report 2
Final Report