Joseph Sin, Molecular Biology
In the US, the American Cancer Society estimates that in 2008, breast cancer killed 40,930 people. Breast cancer remains the most common form of cancer among women today and results in almost 1% of all deaths worldwide. Various types of treatment for breast cancer exist today such as surgery, chemotherapy, radiotherapy, etc. Breast cancer treatments are usually selected based on the specific character of breast cancer: estrogen receptor (ER) positive or estrogen receptor negative. Treatment of breast cancer through estrogen modulation is a common form of treatment. Predicting the overall longer cancer-free survival is best done through measuring the levels of estrogen receptor.
ER is a ligand-dependent transcription factor and is regulated by estrogen binding. More specifically, two subtypes of ER, ERα and ERβ are involved in mediating estrogen functions. ERα binds 17β-Estradiol (E2), the most prevalent form of estrogen. ERα associated with E2 bound to chromatin induces gene transcription by translocating into the nucleus and binds to estrogen receptor element (ERE), a consensus sequence of palindromic repeat (5’GGTCAnnnTGACC3′). ER also recruits cofactor proteins, which results in chromatin remodeling and gene expression regulation through interacting with histone acetylases or transcriptional machinery.
Most studies up till now have focused on how ER activates gene transcription. Recently, estrogen’s repressive effects on target genes were shown. Only within the past five years have we had the necessary advances of the human genome project and microarray technology to realize that ER can also repress gene transcription. The map of the human genome allows scientists to see how ER affects a complete array of genes versus only a limited selection of genes. Microarray technology is a genome wide expression analysis capable of measuring differing gene expression levels. Through comparison of these levels, an analysis of which genes are activated or repressed by ER can be made. A list of 215 putative ER binding sites adjacent to E2-regulated genes within 100kb of their transcriptional start sites suggested that ERα directly represses 66 of those target genes.
Better understanding the mechanisms of ER transcriptional repression would not only increase our understanding of how estrogen affects breast cancer, but also aid in the creation of effective treatments for breast cancer through increased knowledge of what areas of breast cancer cells to target.
My research has two parts. The first part was to find genes that were down regulated by estrogen in order to increase the data pool of genes down-regulated by estrogen. Four target genes, ARGN, MGC16169, CALML5, and NFIB are suspected to be involved in down-regulation by ER. However, after conducting validation tests, these genes were determined to not be repressed. The second part includes characterizing the specific effects of co-repressors NCoR, NRIP1, and SMRT. Removal of these co-repressors and the subsequent effect of their removal on following four ER target sites, HES1, PSCA, SLC35A1, and MME were studied. A knock down of a single co-repressor did not affect the majority of transcriptional activity in ER repressed target genes. A triple knock down was also conducted in hope that removal of multiple co-repressors might affect repression. However, the triple knock down was a failure and future experiments need to be done. Future direction of the project includes repeating the experiment with another set of cDNA. Understanding the mechanisms of ER transcriptional repression would significantly aid the creation of effective treatments for breast cancer.