Discovery Park Undergraduate Research Internship Program

"Landscapes of DNA methylation profile in breast cancer cells upon exposure to dietary polyphenols and their relevance in cancer prevention"

About the Project

Project Time & Type:
Full-Year 2017 - CPIP
Research area(s):
Cancer epigenetics and Nutriepigenomics
Project Description:
Abstract Epigenetic mechanisms have been studied for their role in several diseases, especially cancer. Particularly, DNA methylation patterns provide insight to the activation and silencing of certain genes. Hypermethylation has been found to have a silencing effect on tumor suppressor genes and hypomethylation leads to activation of oncogenes and pro-metastatic genes. To reverse these changes in the DNA methylation patterns occurring in cancer, dietary polyphenols are hypothesized to target gene transcription with abnormal qualities such as hypermethylation and hypomethylation. The use of polyphenols would be of special interest in breast cancer due to high prevalence and epigenetic component of the disease as shown in our previous studies. Using high throughput state-of-the-art technology, the epigenetic effects of a blueberry polyphenol, pterostilbene, in breast cancer will be determined. This project has the potential to show the general public how dietary compounds can play an important role in anti-cancer approaches. Methodology In this study, MCF10CA1a breast cancer cells will be treated with pterostilbene, a polyphenol found in blueberries. In a current study, the proper dosage of pterostilbene to be applied in MCF10CA1a cells will be determined and that dosage will be used throughout the project. The cells will be treated for 4-9 days with pterostilbene followed by cell growth and invasiveness assays. DNA and RNA will be isolated for, respectively, DNA methylation and gene expression analyses. DNA will be subjected to bisulfite conversion and subsequently hybridized to a genome-wide Illumina 450K methylation microarray in order to determine the DNA methylation patterns of untreated cancer cells and cells treated with pterostilbene. The Illumina microarray gives the information about methylation levels at single CpG resolution of 99% of all known human genes. The obtained data will be pre-processed in GenomeStudio software and analyzed in R software using limma package. Twenty genes with the largest statistically significant difference in methylation will be chosen for further analysis and validation of methylation change using pyrosequencing. Bisulfite converted DNA will be amplified in PCR with primers for selected gene regions and pyrosequenced in PyroMark Q24. To rule out cell-specific effects, the results will be compared to another breast cancer cell line, e.g., MCF-7. After the top 20 genes have been identified and validated, QPCR will be conducted to determine if pterostilbene-mediated changes in methylation are correlated to changes in gene expression. We will subsequently perform chromatin immunoprecipitation (ChIP) analyses for validated genes functionally linked to cancer. Using antibodies specific to active RNA polymerase II, the transcription onset of each gene will be determined. The ChIP analysis will be also used to determine the chromatin structure and histone marks such as acetyl H3K9 (associated with activation) and tri-methyl H3K27 (associated with inactivation). Justification and Significance Breast cancer is the most common type of cancer in women and the second most commonly occurring cancer overall worldwide (1, 2). Identification of new effective preventive and anti-cancer strategies is therefore critical. Only 5-10% of breast cancers are hereditary (3, 4). The overwhelming majority of cases are sporadic, likely caused by external exposures including estrogens, alcohol use, physical inactivity, and poor diet (3, 4). It is estimated that at least 30% of sporadic breast cancer cases are not linked to mutations but have been shown to contain epigenetic alterations, particularly in DNA methylation (5, 6). Our preliminary studies indicate that exposure to dietary polyphenols modifies DNA methylation patterns in cancer cells leading to activation of methylation-silenced tumor suppressor genes and to silencing of demethylation-induced oncogenes and other cancer-driving genes. Our goal is to capitalize on dietary polyphenol pterostilbene as a safe agent with the capacity to epigenetically modify gene expression and thus exert anti-cancer effects. Our study will generate novel information for the epigenetic effect of dietary polyphenols. The long-term impact of establishing epigenetic-targeting anti-cancer strategies would be a decrease in cancer incidence, and development of individualized dietary plans for those at risk for cancer. Such findings could also provide support for further studies of the efficacy of the tested polyphenols in other disorders involving epigenetic aberrations (7, 8). References 1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA: a cancer journal for clinicians. 2015;65:5-29. 2. Ly D, Forman D, Ferlay J, Brinton LA, Cook MB. An international comparison of male and female breast cancer incidence rates. International journal of cancer Journal international du cancer. 2013;132:1918-26. 3. Lacey JV, Jr., Kreimer AR, Buys SS, Marcus PM, Chang SC, Leitzmann MF, et al. Breast cancer epidemiology according to recognized breast cancer risk factors in the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial Cohort. BMC cancer. 2009;9:84. 4. Danaei G, Vander Hoorn S, Lopez AD, Murray CJ, Ezzati M, Comparative Risk Assessment collaborating g. Causes of cancer in the world: comparative risk assessment of nine behavioural and environmental risk factors. Lancet. 2005;366:1784-93. 5. Stefanska B, Rudnicka K, Bednarek A, Fabianowska-Majewska K. Hypomethylation and induction of retinoic acid receptor beta 2 by concurrent action of adenosine analogues and natural compounds in breast cancer cells. European journal of pharmacology. 2010;638:47-53. 6. Stefanska B, Salame P, Bednarek A, Fabianowska-Majewska K. Comparative effects of retinoic acid, vitamin D and resveratrol alone and in combination with adenosine analogues on methylation and expression of phosphatase and tensin homologue tumour suppressor gene in breast cancer cells. The British journal of nutrition. 2012;107:781-90. 7. Barres R, Osler ME, Yan J, Rune A, Fritz T, Caidahl K, et al. Non-CpG methylation of the PGC-1alpha promoter through DNMT3B controls mitochondrial density. Cell metabolism. 2009;10:189-98. 8. Scarpa S, Cavallaro RA, D'Anselmi F, Fuso A. Gene silencing through methylation: an epigenetic intervention on Alzheimer disease. Journal of Alzheimer's disease : JAD. 2006;9:407-14.
Expected Student Contributions:
The intern would be involved in isolating RNA and DNA from breast cancer cells and mammary epithelial cells treated with pterostilbene; processing samples for genome-wide DNA methylation analysis by Illumina 450K; assessing DNA methylation of candidate genes by pyrosequencing; establishing expression of candidate genes by QPCR; performing functional analyses such as cell growth, invasion and soft agar assays. He/she will participate in data analyses including processing genome-wide data in GenomeStudio software; creating charts in Excel and presentation in ppt; discussing the results and writing a manuscript/report.
Related Website(s):
http://epigenetics.wix.com/stefanskalab
Desired Qualifications:
At least 3.4 GPA
Estimated Weekly Hours:
10
Department awards independent research credits for this project?
Yes, 3 credit hours

Professor in Charge

Name:
Stefanska, Barbara
Deptartment/College:
Nutrition Science

Student Supervisor

Name:
Barbara Stefanska
Title:
Assistant Professor

Cooperating Faculty

Name:
Sophie Lelievre
Deptartment/College:
Basic Medical Sciences