Laura Castillo and Dr. Chin-Yo Lin, Department of Microbiology and Molecular Biology
Estrogen plays a role in the reproductive maturation of females, including breast development. It is also known that estrogen affects the development of malignant breast tumors. Estrogen works by binding to specific estrogen receptors found in the cytoplasm or near the nuclear membrane. Once bound to estrogen, these receptors diffuse into the nucleus where they bind to specific DNA sequences called estrogen-response elements. They then regulate the expression of specific genes. There are two types of estrogen receptors (ER): ERα and ERβ. Only ERα has been observed to play a role in breast cancer development. Dr. Chin-Yo Lin and associates used large-scale microarray experiments to characterize the specific genes regulated by ERα in breast tumor cells. Out of 19,000 genes, 137 were observed to be either inhibited or activated by ERα.
In this project, I attempted to study the effects of three out of the 137 genes identified by Lin, et al. on breast cancer cells, namely IL6ST (Interleukin 6 signal transducer), IGSF4 (Immunoglobulin superfamily, member 4), and DNAJC12 (DNA J Hsp40 homolog subfamily C, member 12). The products of these three genes have been associated with various forms of cancer, including choriocarcinoma (cancer in the uterus that develops during ectopic pregnancies), cervical cancer, and prostate cancer. IL6ST and IGSF4 have also been observed to play a role in cell growth or cell death, cellular functions highly associated with the development of cancer.
The primary step of this project was to validate the up-regulation of IL6ST, IGSF4, and DNAJC12 by estrogen. This was necessary because microarray analyses involve very large number of genes, increasing the probability of obtaining false positives or false negatives. To do so, I used the human breast cancer cell line MCF-7. The cells were treated with either estradiol, a form of estrogen, or with ethanol. The cells treated with ethanol were used as a control, since ethanol should cause no change in gene expression. The cells were then allowed to grow for 24 hours under the experimental conditions.
RNA was extracted from the cells by lysing the cells and then purifying the RNA by affinity chromatography. The concentration of RNA was measured using a nanodrop machine and its quality determined by agarose gel electrophoresis and ethidium bromide staining. cDNA was then made from the RNA samples by using PowerScript Reverse Transcriptase.
The cDNA samples were analyzed by Real-Time (RT) PCR using primers specific for each gene (Table 1). These primers were designed using Primer3 (available at http://fokker.wi.mit.edu
/primer3/input.htm) and ordered from MWG Biotech. RT-PCR works by quantifying the number of gene copies after each amplification cycle using fluorescent reporter probes. A PCR thermocycler measures the fluorescence emitted by the genes as they are amplified, corresponding to the increase in product. This determines the gene’s CT (crossing threshold); a smaller CT indicates that the gene was present in the sample at a greater concentration. The controls used were beta-ACTIN, a gene that should show no change with estrogen treatment, and GREB1, a gene known to be up-regulated by estrogen. Each gene was analyzed in duplicate. The results were expressed as n-fold change in gene expression of the genes of interest and the positive control GREB1 relative to the expression of beta-ACTIN. The relative expression was calculated using the Delta Delta CT formula (2-ΔΔCT = ΔCT) where CT = the fluoresence threshold value given by the thermocycler. The CT of the target gene (untreated) is subtracted from the CT of beta-ACTIN (untreated), and the CT of the target gene (treated) subtracted from the CT of beta-ACTIN (treated). Finally, the treated ΔCT value is subtracted from the untreated ΔCT value, giving as a result the n-fold change of the gene in the estrogen-treated cells. All genes were shown to be up-regulated by estrogen: IL6ST by 3.84, IGSF4 by 2.13, and DNAJC12 by 2.90 fold (Figure 1).
The results for DNAJC12 are supported by a recent publication; in addition, this study indicates that the gene’s behavior in response to estrogen is correlated to breast cancer development. With regards to IL6ST and its behavior in breast cancer, there are various contradicting reports; while some indicate that its presence in breast carcinomas cause apoptosis of cancerous cells, other’s disagree and show that the gene increases proliferation of malignant cells. Moreover, none of these studies focus on IL6ST’s response to estrogen. The gene IGSF4 has been shown to be significantly up-regulated in malignant tissues, but its behavior in breast cancer cells, especially with relation to estrogen has not been thoroughly studied.
The results in this study and others indicate that research on the effects of IL6ST and IGSF4 in response to estrogen in breast cancer cells should continue. Students in Dr. Chin-Yo Lin’s lab will proceed to knock-down the IL6ST and IGSF4 genes from MCF-7 cells and use cell proliferation assays to determine their effects on breast tumor development as it was done with DNACJ12. It is expected that the final results will contribute valuable knowledge that will aid in developing treatments to prevent and cure breast cancer.