a. HPV oncoprotein associated host cellular proteins The HPV E6 and E7 protein exert their carcinogenic activities by associating with and functionally reprogramming host cellular signal transduction protein complexes. We are using proteomic approaches to isolate and identify components of these complexes and are investigating the biological consequences of these associations. b. HPV induced genomic instability Genomic destabilization is a key process in cancer development. Expression of HPV16 E6/E7 oncoproteins in primary human epithelial cells causes genomic instability. Centrosome associated mitotic abnormalities. Centrosomes form the mitotic spindle poles and we have shown that HPV16 E7 expression causes aberrant centriole synthesis. We are investigating the molecular mechanisms of this process. Formation of supernumerary centrosomes, however, is not sufficient for formation of multipolar mitosis due to a cellular defense mechanism, centrosome coalescence, whereby supernumerary centrosomes form a single mitotic spindle pole. We know that co-expression of E6 and E7 gives rise to multipolar mitoses and we are investigating whether and how E6 subverts centrosome coalescence. Lagging chromosomes. We observed an increased incidence of mitoses with chromosomal material that does not move to the mitotic plate. Our recent work revealed that HPV16 E7 expressing cells exhibit a prometaphase delay that we have linked to association with the Nuclear and Mitotic Apparatus protein 1 (NuMA), which in turn leads to delocalization of dynein from mitotic spindles presumably affecting its motor activity. Because these proteins also affect mitotic spindle orientation, we are interested in determining whether this affects mitosis of basal epithelial cells in 3-dimensional “raft” models. Double strand DNA break repair: HPV oncoprotein expressing cells In addition, E7 expressing cells also exhibit evidence for increased double strand DNA breaks, which causes structural chromosomal aberrations. We are investigating the biochemical bases for these genome-destabilizing activities of HPV oncoproteins. c. Identification of therapeutic targets of HPV associated disease and cancer We collaborate with Ed Harlow’s group at the Harvard Institute of Proteomics (HIP) to identify protein kinases that exhibit a “synthetic lethal” phenotype in cervical carcinoma cells. The hypothesis of these studies is that HPV oncogene expression in a normal human epithelial cell causes significant physiological alterations (“oncogene addiction”) that can be exploited to identify specific synthetic lethal phenotypes and that we will be able to assign these phenotypes to specific activities of the HPV oncoproteins. In the course of these studies we have identified 2 kinases that score as synthetic lethals with loss of p53 activity, ie RNAi mediated depletion of these kinases causes cell death in cells with defective p53 activity whereas is does not markedly affect cell growth/survival in normal cells. We have performed a small molecule inhibitor screen for one of these kinases and are evaluating hit compounds. d. “Systems biology” approach to globally investigate HPV/host cell interactions We have initiated an effort to globally capture the biochemical intersection of viral and host protein “interactome” networks using yeast genetic and biochemical techniques and to determine the transcriptional consequences of the dynamic changes of virus/host cell interactions. This work is done in collaboration with Marc Vidal at the Dana Farber Cancer Institute. These studies will allow us to integrate the data sets with genetic mapping information, predicted and established gene functional role classifications and metabolic pathway assignments, phenotypic classifications, metabolic profiling patterns, and clinical data and holds the promise to arrive at a comprehensive and integrated view of HPV/host cell interactions. e. HPV-induced alterations of epigenetic programs. We discovered that HPV16 E7 can associate with the non-canonical E2F family member E2F6. Unlike other E2F family members, E2F6 is not regulated by retinoblastoma protein family members but is a component of polycomb repressive complexes, in particular the “maintenance complex” PRC1. PRC1 binds to H3K27me3 modified, silenced chromatin and we have shown that detection of E2F6 containing PRC complexes is greatly diminished in E7 expressing cells. We are now investigating expression of H3K27me3-specific demethylases and since polycomb group proteins regulate expression of HOX genes we are currently evaluating whether HOX genes are differentially expressed in E7 expressing versus normal cells. f. HPV-associated metabolic alterations. Our results indicate that HPV16 E7 expressing cells may suffer from metabolic stress and are undergoing an autophagy related process. We have shown that expression of the HPV16 E6 protein may balance this effect and keep the cells in a state of "blissful ignorance". Consistent with this model we have shown that HPV16 E6 causes sustained mTORC1 activation even under conditions of nutrient deprivation. We are investigating the molecular mechanism of mTORC1 activation by HPV16 E6. Back |