We are interested in identifying and characterizing novel genes and mechanisms involved in the development of cancer. To achieve these goals we have chosen three key areas of study, 1) the two central pathways in cancer (pRB and p53 pathways), 2) the role of chromatin modifiers in cancer and 3) the molecular mechanisms regulating genomic stability. We are using several different experimental approaches to achieve our objectives, including phenotype screenings using small hairpin (sh) RNA libraries, target gene identification by gene expression profiling and chromatin immunoprecipitations, determination of expression patterns in primary human tumors using tissue microarrays, generation of knockout mice with deletion of specific genes, and identification of novel proteins and protein complexes by yeast two-hybrid screening and protein purification followed by mass spectrometry. Recently, we have also introduced a number of functional genomics screens designed to identify novel targets for cancer therapy. By performing this work, we expect to identify novel genes and to reveal mechanisms involved in the development of human cancer. Moreover, we expect that some of these newly identified genes will be excellent targets for the development of novel drugs for cancer therapy.Major ongoing projects
1. Functional role of the E2F transcription factors in cell proliferation and cancer
The E2F transcription factors are the main downstream effectors of the retinoblastoma protein (pRB) pathway of which the alteration is an obligatory event for the development of most, if not all, human cancers. Due to the key role of the E2F transcription factors in normal and neoplastic growth, it is important to understand the biological roles of the E2Fs and the mechanisms by which they regulate transcription. Currently, we are working on projects related to the two most newly identified E2Fs, E2F6 and E2F7. We have generated mice with the deletion of two E2F6 interacting proteins, Epc1 and Epc2. Furthermore we are analyzing the functional consequences of knocking out the putative tumor suppressor E2F7. Several other projects related to the E2Fs are ongoing in the laboratory, including studies of the role of E2F in cell proliferation, the genetic requirements for E2F1 induced apoptosis and the role of E2F3 induced senescence in vivo.
2. The role of newly identified E2F regulated genes in cell cycle progression and cancer
Over the last five years we have performed several screens to identify novel target genes for the E2Fs. We have been interested in identifying such genes, since we believe that it is essential for understanding the regulation of cell cycle progression. In addition, the E2F target genes are putative oncogenes and tumor suppressor genes. Our strategy consists of three successive steps: i) identification of novel E2F target genes ii) selection of those with a potential role in tumorigenesis using bioinformatic tools and/or tissue microarrays iii) characterization of their role in cell proliferation/tumorigenesis and elucidation of their molecular function. By using this strategy we have identified several genes, which are overexpressed in a large number of human primary tumors. One of these genes is the Polycomb Group (PcG) gene, EZH2, whose overexpression correlates with aggressive breast and prostate cancer. Currently, we are working on five additional E2F target genes, which we have found overexpressed and in some cases genetically amplified in primary human cancer. Our aims are to understand whether they are bona fide oncogenes, to elucidate their biochemical and biological function, and to investigate if they have diagnostic or prognostic value for cancer treatment.
3. The role of Polycomb group genes in cell proliferation and cancer
Polycombs (PcGs) are best known for their role in maintaining the expression of the HOX genes during development, and the deletion of single PcGs result in homeotic transformation during embryogenesis and misexpression of HOX genes. Several lines of evidence now suggest that PcGs have a role in regulating normal proliferation and that deregulation of their expression can result in cancer. This project has two main objectives. The first is to understand how the PcG complex, PRC2 containing EZH2, EED and SUZ12 regulates cell proliferation and how its deregulation contributes to cancer. The second is to use functional genomics approaches to identify novel interactors of the PcG proteins and to characterize their role in normal cell growth and cancer.
4. Checkpoint mechanisms involved in the DNA damage response
Genomic instability and insensitivity to DNA damaging agents and DNA replication errors are believed to be essential for the development of human cancer. For a number of years we have investigated the mechanisms regulating the initiation of DNA replication in mammalian cells. Recently, we have shown that Geminin is required for preventing rereplication and maintaining genomic stability in mammalian cells. Currently we are performing several functional screens (see below) to identify novel genes, which are essential for a normal checkpoint response. Furthermore, specific projects are ongoing, which analyze the potential tumor suppressive properties of novel p53 regulated genes.
5. Functional screens to identify novel target genes for cancer therapy
The recent discovery that small interfering RNAs can specifically prevent the synthesis of target genes in mammalian cells has been a major revolution for molecular biologists. We are now able to specifically block the expression of specific genes and measure the effect on cell proliferation and tumor development. The subsequent development of vector systems that allow the stable expression of small hairpin RNAs has introduced an experimental tool to screen for phenotypes incurred by loss of function of specific gene products. As part of a major effort, in part financed by the EU framework 6 program, we are currently using retroviral shRNA libraries and retroviral insertion mutagenesis to screen for genes that affect specific cellular phenotypes associated with cancer. We anticipate that these screens will identify several novel tumor suppressors and oncogenes. Once identified we will determine if these genes are either lost or overexpressed in human tumors, and we will elucidate their role in oncogenesis.