Loss of PATCHED (wechs-affy-mouse-512645)

We are examining the genes that control initiation and progression of murine medulloblastomas that result from loss of patched. Approximately 25% of human medulloblastomas have mutations in patched or in other elements of the sonic hedgehog pathway. However, the cells from which these tumors originate (neural progenitors or stem cells), the cells that are responsible for tumor propagation (cancer stem cells), and the genes that are required for tumor progression are poorly understood. To address these questions, we have developed conditional patched knockout mice in which the gene is deleted in neural stem cells or progenitors. In addition, we have isolated a population of tumor-propagating cells from these tumors. By studying these models we will gain insight into the mechanisms of tumorigenesis and identify new targets for therapy. To identify the molecular mechanisms that underlie tumor initiation and propagation, we will compare gene expression in tumors resulting from loss of patched in neural stem cells vs. progentors, and in cancer stem cells vs. non-cancer stem cells. We hypothesize that medulloblastomas initiated in neural stem cells (using GFAP-Cre) are more aggressive than those initiated in neural progenitors (using Math1-Cre), and will express more genes associated with self-renewal. In addition, we hypothesize that cancer stem cells (CD15+ cells) from a given tumor will express higher levels of self-renewal genes than non-stem cells (CD15- cells) from the same tumor. Cells from 5 GFAP-Cre tumors, 5 Math1-Cre tumors, and 5 conventional patched-knockout tumors will be isolated using enzymatic digestion and Percoll gradient centrifugation. These procedures have been found to result in 85-95% pure tumor cell populations. In addition, 5 tumors will be FACS-sorted to isolate CD15+ and CD15- populations. RNA will be prepared from each of these samples using an RNeasy kit from Qiagen. Samples of 2 micrograms will be resuspended in 10 microliters of RNase-free water, and sent to the Consortium for labeling, hybridization and analysis. Cells from the three types of tumors, and CD15+ and CD15- cells, will be compared to one another.