Question

In a recent study, the expression profiles for 6900 genes in normal and malignant breast tissues from rats were compared. RNA was extracted using the following tissues: a. breast tissue from virgin rats b. breast tissue from pregnant rats c. breast tissue from lactating rats d. breast carcinomas induced by the meat-derived carcinogen, PhIP e. breast carcinomas induced by the experimental carcinogen, DMBA Tissues a, b, c were grouped together as normal and tissues d, e were grouped as carcinoma samples. Genes that were either induced (more than 2-fold increase in expression) or repressed (more than 2-fold decrease in expression) in both carcinomas were determined compared to normal samples. A partial listing of differentially expressed genes is below: Induced: Cell growth and cell cycle related genes -platelet derived growth factor A (PDGF-A) -cyclin dependent kinase 4 (CDK4) -cyclin D Signal transduction and transcription related genes -FOS -MYC - Rho-A Repressed: Extracellular matrix genes alpha 1 type v collagen -actin -fibronectin Signal transduction and transcription related genes -p53 What was the purpose of analyzing breast tissue from pregnant and lactating rats? b) What characteristic of cancer is stimulated by the overexpression of PDGF4, Cd K4, and cyclin D? Mention factors involved in different stages of the cell cycle c) What specific characteristic of cancer might be stimulated by the 1) overexpression of Rho-A? 2) the repression of extracellular matrix genes? d)FOS and MYC are transcription factors that regulate the expression of cyclin D, Bcl-X, and other genes. Why is it possible for these carcinomas that no mutation happens in the cyclin D gene, despite its overexpression? )Would you anticipate greater or fewer differences in gene expression if two different types of cancer induced by the same carcinogen were compared? f) p53 is a transcription factor that promotes the transcription ofa CdK inhibitor in cells with damaged DNA. Explain why the levels of p53 are repressed. Mention stages of the cell cycle.

Answer 1

a. Genes involved in breast cancer are identified by the study. Mammogenesis occurs during pregnancy and lactation for milk production, under influence of estrogen, progesterone and prolactin. This mammogenesis cause increased cell proliferation, which is not cancerous. In breast cancer too there is proliferation of breast cells. However, this proliferation is abnormal and also induced by estrogen. Estrogen induced breast cancer is metastatic and can spread. Comparison of breast tissue from cancer patient and pregnant/lactating rats, will identify only specific genes involved in breast cancer. Thus, lactating and pregnant rat tissue are control tissues for the experiment.

b. There is no PDGF4. Instead it is PDGFA. PDGFA is platelet derived growth factor involved in cell cycle. Increased PDGFA causes increased cell division, leading to cancer. PDGFA binds its tyrosine kinase receptors on breast tissue, causing tyrosine phosphorylation and activation of downstream signaling that leads to proliferation. PDGFA is involved in G1-S phase transition. CDK4 interacts with cyclin D in G2 phase and is involved in phosphorylation of retinoblastoma gene. Overexpression of Cyclin D and CDK4 allow the cells to remain in G1 phase.

The abberant genes involved in cell cycle are p53, myc, FOS, and RhoA. P53 is a tumor suppressor gene that inhibits cells cycle and induces apoptosis. Myc is a proto-oncogene that causes increase progression through G1 and G2 phases. FOS is an AP- transcription factor, which is an oncogene. RhoA GTPase id indirectly involved in cell cycle as it is an oncogene.

c 1) When RhoA is overexpressed, it causes the breast cancer cells to proliferate, migrate and adhere to other tissues. Thus, it induces metastasis in cancer cells, where in cancer cells will migrate to other tissues and cause cancer of those tissues. Interaction of RhoA with ROCK is required for cell migration.

2) Extracellular matrix proteins are involved in ell growth, migration and survival. Downregulation of these ECM proteins induce EMT (epithelial mesenchymal transition) causing increased cell migration, promoting metastasis. Collagen and fibronectin allow attachment to basement membrane while actin filaments form the cytoskeleton.

d. FOS and myc are transcription factor that bind to promoter of cyclin D. FOS binds to the AP-1 site in the promoter situated at -903 position. Myc acts by hyper-phosphorylating Rb protein. It also binds cyclin D promoter and activates it. As the increased expression of cyclin D is due to increased protein synthesis, no mutations will be seen in cyclin D. This is translational regulation by myc and Fos.

e. Mechanism of cancer progression will depend on genes expressed in the tissue. Suppose, there is no activation or expression of Ras oncogene in the tissue. Then the carcinogen that activates Ras will not act on the tissue. If the tissues expressed ER, then only estrogen will act as carcinogen in that tissue. Thus, skin show no effect of estrogen while estrogen will induce breast and ovarian cancer. The tissues that express the target gene (ovary and breast) will show fewer differences in genes. Greater differences in expression of genes in response to estrogenic carcinogens will eb seen between skin and breast.

f. Tumor suppressor genes inhibit cell cycle via apoptosis. If p53 expression (a tumor suppressor protein) is reduced, apoptosis is inhibited. P53 induces expression of CDK inhibitor p21WAF1/CIP1, GADD5 etc, resulting in increased expression of CDK2-cyclin E as well as CDC2-Cyclin B interaction. This will inhibit progression to S and M phase. Reduced p53 expression will therefore allow increased cell division. Hence, in breast cancer, p53 expression is reduced.