Progression from a non-transformed normal cell to a malignant cancer cell requires multiple genetic changes that activate oncogenes while restraining tumor suppressors. This occurs via the acquisition of genetic mutations and changes of gene copy number. Whole chromosome and segmental aneuploidy can alter copy number of relevant oncogenes and tumor suppressors and perturbation of the machinery controlling the fidelity of chromo- some segregation during mitosis is sufficient to promote tumor development via the generation of aneuploidy. The HECT-domain ubiquitin ligase HUWE1 coordinates the proteasomal-mediated degradation of proteins with crucial roles in cell division, differentiation and survival and recent work has shown that inactivation of HUWE1 contributes to tumor development. Initially, we generated and studied conditional knockout mice in which HUWE1 inactivation was targeted to the nervous system. We used this mouse model to characterize the role of HUWE1 in neurogenesis. We found that the activation of MYCN oncoprotein and MYCN-dependent transcriptional net- works are crucial events operating downstream of HUWE1 and whose de-regulation leads to developmental aberrations in the HUWE1-null brain. We also determined that genetic and epigenetic mechanisms drive loss- of-function alterations of HUWE1 in a significant fraction of human brain tumors. More recently, our work has revealed novel and unexpected functions of HUWE1 that have defined this ubiquitin ligase as crucial regulator of mitotic fidelity whereby loss of HUWE1 induces chromosome mis-segregation and an unstable genome. The present proposal embodies three Specific Aims that will investigate the novel functions of HUWE1 and will serve as entry point into the tumor suppressive mechanisms of HUWE1.
In Aim 1 we will investigate the events that are controlled by HUWE1 to preserve mitotic fidelity and genome integrity.
Aim 2 will examine the role of candi- date HUWE1 substrates that we have identified through unbiased proteomic methods for the accumulation the mitotic phenotype initiated by loss of HUWE1 activity.
In Aim 3 we will proceed to address the full scope of genetic lesions that cooperate with the accumulation of DNA damage and the mitotic phenotype triggered by HUWE1 deletion to initiate brain carcinogenesis using the Sleeping Beauty transposase insertional mutagenesis.
Abnormal mitosis is one of the main mechanism that generates aneuploidy (changes in chromosome number) and chromosomal instability, two hallmarks of cancer cells. Goal of this proposal is the dissection of the pheno- typic and biochemical events engaged by the ubiquitin ligase Huwe1, a tumor suppressor protein that has emerged from our work as master regulator of genome stability in normal cells.