Team:METU HS Ankara/Description
Project Motivation
Colon cancer is a widespread disease which is the fourth most common cancer type throughout the world. (National Cancer Institute, 2016) After, reading numerous articles about cancer types, we focused on colon cancer as it is directly related to alimentation and exercising.In colon cancer, polyp formation is observed. They are abnormal growths in colon’s inner surface. Polyps are benign (non-cancerous) growths, but cancer can start in some types of them; like Adenomatous polyps.
http://hcpthink.com/mpanel_document/liquid-biopsy-can-predict-colon-cancer-recurrence/
In Turkey 7 in 100,000 is suffering from colon cancer and each year 3200 people die from colorectal cancer. (Tatar&Tatar, 2010) Although Turkish people’s unhealthy diet differs depending on the region they live and socio-economic status, in general their diet are composed of more meat and less vegetables. (Hacettepe University, Faculty of Health Sciences, Nutrition and Dietetics Department, 2014)
Being obese, physical inactivity, certain types of diet, smoking, using alcohol greatly increase the risk of developing cancer (American Cancer Society, 2015). In order to prevent colon cancer, firstly you need to be aware of healthy lifestyle. On top of that, Turkish people can’t find time to exercise regularly. Also the preferred transportation way is cars, so people tend to move less. A non-fiberous diet with too much red meat plus insufficient amount of exercise can lead to cancer especially to colon cancer. Because of these alarming facts we decided to work on this topic as every contribution counts in fight against cancer.
Though treatment is available in forms of chemotherapy, surgery and targeted drug therapy they also include various side effects (MayoClinic, 2016). Our first motivation was 2015 Harvard Biodesign Team’s successfully targeted mechanism that give us an opportunity to create and form a micro-environment. Secondly, we decided to find an alternative solution consist of an endogenous substance so that there will be no side effects, best candidate was butyrate. The research about butyrate’s effects on colon cancer has been done by Dr. Sreeparna Banerjee’s lab from Biological Science Department at METU, also encouraged us to focus on Butyrate.
Defining The Problem
Our project handle two main questions. First question was, “Is the colon cancer formation correlated with unhealthy diet such as non-fiberous diet and increase in consumption of carbohydrates?” Second one is,”Does the reduction of butyrate producing bacteria directly or indirectly lead to colon cancer formation?” and we have designed our project accordingly to get answer to these questions. Based on our collaborative work with research from Biological Science Department at Middle East Technical University, we had an opportunity to test our design with co-culture on cancer cells. Our challenge was then to develop a method to detect cancer, form a micro-environment and inhibit cancer formation.
Why Butyrate?
At the beginning of our project, we recorded a few proteins fitting our goal like TSP1, VASH1 ,A4.6.1 , 17-1A and vitamin D.
When we inquired about those proteins we saw that their arrangements are too long to be expressed in E. coli. Since they are eukaryotic (have introns, needs a further protein modification that cannot be performed in a prokaryotic cell and also we can’t be sure if they are folded accurately or not) and not found naturally in colon, we then started to search an organic compound found naturally in colon and prokaryotically synthesized by the help of bacteria. The best candidate was the butyric acid.
Butyrate is a short chain fatty acid which is formed by bacterial fermentation of carbohydrates (Sengupta et al., 2005). We found out that butyrate prevents colon cancer by promoting differentiation, cell-cycle arrest and apoptosis of transformed colonocyte (Hassig et al., 1997). Since butyrate is already present in the colon it differs from other cancer therapies by not having side - effects (when the concentration is accurately arranged). There are many pathways to produce butyrate. Unlike Toulouse 2015 iGEM Team, we didn’t want to use so many enzymes, because it would create product inefficiency and a crowd in bacteria. We decided to work with a shorter pathway. As Acetyl CoA is readily present in the cytosol and it can be directly converted into our agent, Butyrate, the best choice for us. This enzyme is called ButCoAT (butyryl coA:acetate coA transferase).
The alternative pathways to produce butyrate can be seen below;
Why FimH?
Last year, Team Harvard 2015 developed a way for E. coli to bind to cancerous cells in the colon. They used pathogenic E. coli. It has a structure called “Type 1 pilli”, the hairlike appendages on the surface of the bacteria which is expressed from “Fim” gene system. (Harvard BioDesign, 2015) At the end of the pili there’s a protein called “FimH” from Fim family which binds naturally to sugar mannose on the epithelial cells. (Sauer et al., 2016) As we want to make our bacteria to bind colon tumor cells, we had to choose a tumor specific binding peptide. We decided to work on “RPMrel”, a colon tumor specific binding peptide, likewise Harvard Bio Design 2015 iGEM team. (Kelly & Jones, 2003)
This year, we will take their project to the next level; treatment. Our E. coli will bind to the cancerous cell and after binding create a microenvironment and it will start producing butyrate, which will induce apoptosis in the cancerous cell.
How will our project work?
Our main goal in this project is to eliminate colon cancer by using our genetically engineered E. coli (nonpathogenic laboratory strain) to bind to cell surfaces with the help of site directed mutated FimH with RPMrel to bind specifically to cancer cells. After binding phase, our bacteria will secrete butyrate which induces apoptosis.
References:
1. Tatar, M., Tatar F. (2010). Colorectal Cancer in Turkey: Current Situation and Challenges for the Future
2. American Cancer Society. (2015). Colorectal Cancer risk factors. Retrieved from: http://www.cancer.org/cancer/colonandrectumcancer/detailedguide/colorectal-cancer-risk-factors
3. Hacettepe University, Faculty of Health Sciences, Nutrition and Dietetics Department. (2014). Beslenme Durumu ve Alışkanlıklarının Değerlendirilmesi Sonuç Raporu
4. Mayo Clinic Staff. (2016). Colon Cancer. Retrieved from: http://www.mayoclinic.org/diseases-conditions/colon-cancer/diagnosis-treatment/treatment/txc-20188274
5. Sengupta, S., Muir, G.J., Gibson, P. R. (2005). Does Butyrate Protect from Colorectal Cancer?
6. Hassig, C.A., Tong, J.K., Schreiber, S.L. (1997). Fiber-derived Butyrate and the Prevention of Colon Cancer
7. Harvard Biodesign. (2015). Retrieved from: https://2015.igem.org/Team:Harvard_BioDesign
8. Sauer MM, Jakob RP, Eras J, Baday S, Eriş D, Navarra G, Bernèche S, Ernst B, Maier T, Glockshuber R. (2016 March 7). “Catch-bond mechanism of the bacterial adhesin FimH” Retrieved from: http://www.ncbi.nlm.nih.gov/pubmed/26948702
9. Kelly, K. A., Jones, D. A., (2003). Isolation of a Colon Tumor Specific Binding Peptide Using Phage Display Selection
9. National Cancer Instute. SEER Stat Fact Sheets: Colon and Rectum Cancer. Retrieved from: http://seer.cancer.gov/statfacts/html/colorect.html
