Embryonal carcinoma (EC) are the stem cells of testicular germ cell tumors (TGCTs), are pluripotent, and share remarkable genetic and biologic similarity to human ES cells. We contend that EC can be seen as an archetypal model of successfully treated, malignant cancer stem cells. Our efforts to define the molecular mechanisms of retinoic acid (RA) mediated tumor cell differentiation using global gene expression analysis and a unique system of RA-sensitive and RA-resistant human EC has led to our hypothesis that a critical determinant of loss of tumorigenicity of EC is epigenetic reprogramming. This evidence is based in part on a set of candidate pluripotency genes, including a polycomb group gene involved in Hox gene repression, PHC1/EDR1, that we discovered to be rapidly downregulated by RA at the lineage commitment window. We provide evidence that EDR1 and other pluripotency genes may be regulated by RA via RA-mediated downregulation of the master pluripotency factor Oct4. Interestingly, many of these RA-downregulated pluripotency genes reside on chromosome 12p, which is uniformly amplified in TGCTs. Hence, we propose that the malignant conversion of the normal primordial germ cell to the EC cell may involve """"""""enforced"""""""" pluripotency with associated chromatin remodeling. The hypothesis to be tested is that RA-induced loss of stem cell renewal is in part due to transcriptional modulation of EDR1 leading to epigenetic reprogramming and lineage commitment of EC cells. The goal of this R21 Stem Cells and Cancer application is to begin to characterize histone modifications in EC cells associated with tumorigenic and curative potential. Our focus is the polycomb complex since alterations in this complex are known to be closely associated with a variety of human cancers and since we have shown that RA downregulates a specific polycomb gene EDR1 that is localized to the critical 12p amplicon in TGCTs. This will be accomplished through molecular, genetic and cell biologic approaches to complete the following specific aims. 1. To define polycomb-specific chromatin modifications of RA-sensitive EC and RA-resistant EC and to determine whether RA is a major morphogenic signal regulating polycomb-specific repression and cancer stem cell renewal. 2. To determine the role of the RR12 gene, EDR1, in RA-mediated, polycomb-specific reprogramming of cancer stem cell renewal in embryonal carcinoma. These studies are important because the unique """"""""germ cell-like"""""""" epigenetics of human EC may be linked to the curability of TGCTs. Testicular germ cell tumors (TGCTs) are one of the few solid tumors curable with chemotherapy even when highly advanced, suggesting that the self-renewing, cancer stem cells of TGCTs are likely targeted during therapy. Completion of the aims of this grant will increase understanding of the genetics and biologic progression of TGCTs and may increase understanding of why these tumors are so curable when the vast majority of advanced solid tumors are not. This in turn may lead to new therapies that effectively target crucial cells within tumors, cancer stem cells. ? ? ? ?
