Date of Award

January 2012

Document Type

Open Access Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Tobias Carling

Subject Area(s)

Surgery, Genetics

Abstract

EPIGENETIC EVENTS SUGGEST A DISTINCT MOLECULAR PATHOGENESIS IN BRAF-ASSOCIATED PAPILLARY THYROID CANCER.

Ogechukwu Eze, Lee Starker, William Long, Robert Udelsman, Peyman Björklund, and Tobias Carling. Department of Surgery, Yale University School of Medicine, New Haven, CT, USA.

Papillary thyroid cancer (PTC) associated with a somatic V-raf murine sarcoma viral oncogene homolog B1 (BRAF) V600E mutation has been associated with a more aggressive phenotype (i.e. extrathyroidal extension, lymph node metastasis, high TNM stage and recurrence) than those with wildtype (WT) BRAF. The underlying molecular mechanism for this association is incompletely clarified. Epigenetic alterations, such as DNA methylation participate with genetic abnormalities to cause altered patterns of gene expression and/or function leading to oncogenesis. Using a quantitative, genome-wide approach to evaluate the PTC DNA methylome, we identified a distinct DNA methylation profile in BRAF-associated PTC. This represents the first, unbiased, systematic, quantitative genome-wide evaluation of DNA methylation alterations in PTC.

A distinct DNA methylation profile was noted in BRAF V600E PTC versus BRAF WT PTC. Twenty-four genes were significantly hypermethylated in BRAF V600E PTC versus BRAF WT (p<0.005 for all). Genes frequently and significantly hypermethylated in BRAF V600E PTC included those involved in transcriptional regulation and cell cycle control such as CDKN2B/p15, RASSF1A and CD6. 25 BRAF V600E PTC and 25 BRAF WT PTC were used for validation of the methylation profile of RASSF1 and analysis of its gene expression. One of two CpG islands in the RASSF1A promoter was differentially methylated with an average of 73% and 8% methylation in BRAF V600E PTC and BRAF WT PTC respectively. In addition, 92% (n=23) of the BRAF V600E tumors were hypermethylated at this CpG island, compared to 12% (n=3) of the BRAF WT tumors. Expression of RASSF1A was decreased in BRAF V600E PTC relative to expression in BRAF WT PTC.

The DNA methylation profile of PTC correlates with BRAF mutational status, underscoring its importance in PTC development. The molecular mechanism of the more aggressive phenotype of BRAF V600E PTC may be related to aberrant DNA methylation of genes involved in transcriptional and cell cycle control. Functional studies assessing the effect of azanucleoside, a demethylating agent on RASSF1A gene expression may further determine a cause-effect relationship. Furthermore, conducting such functional experiments in BRAF V600E PTC and BRAF WT PTC cell cultures would be critical in assessing the possibility of clinically applying a demethylating agent to treatment of aggressive, BRAF V600E PTC.

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