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The Wistar Institute

3601 Spruce Street

Room 308

Philadelphia, PA 19104

Office: 215-495-6840

rzhang@wistar.org

Epigenetics, Cellular Senescence and Tumor Suppression

Introduction and overview of research interests

The ultimate goals of our research are to:

1) understand the basic mechanisms underlying oncogene-induced senescence, an important tumor suppression mechanism in vivo, and

2) identify novel intervention strategies for epithelial ovarian cancer (EOC), the most lethal gynecological malignancy in the developed world.

Cellular senescence is a state of irreversible cell growth arrest. In primary mammalian cells, cellular senescence can be triggered by activation of an individual oncogene (such as RAS). This process is termed oncogene-induced senescence. Oncogene-induced senescence is an important tumor suppression mechanism in vivo, driving irreversible growth arrest of cancer progenitor cells that harbor an initial oncogenic hit. However, the definition of an oncogene is a gene that actively promotes tumorigenesis. We are interested in the molecular mechanism that reconciles the tumor-promoting nature of oncogene activation with the tumor-suppressing role of oncogene-induced senescence. Notably, oncogene-induced senescence is often characterized by aberrant DNA replication, which is thought to drive accumulation of DNA damage. As a new direction in the lab, we are investigating the molecular mechanism underlying oncogene-induced aberrant DNA replication and its contribution to the senescence-associated cell growth arrest.

EOC represents the fifth leading cause of death from cancer among women. Thus, there is an urgent need for novel therapeutic approaches for this devastating disease. To develop such approaches, we must better understand the etiology of the disease. The genetic alterations that characterize EOC are known to induce senescence. However, the role of cellular senescence in EOC development remains poorly understood. We are investigating the role of Wnt signaling in regulating senescence of EOC cells. Specifically, we are interested in the role of Wnt5a, a non-canonical Wnt ligand, in promoting senescence of EOC cells via epigenetic regulators. In addition, compared with our extensive understanding of the genetic abnormalities in EOC, epigenetic dysregulation of gene transcription in EOC remains less well studied beyond an initial characterization of DNA methylation. Polycomb proteins modify chromatin such that epigenetic silencing of genes takes place. Elevated levels of Polycomb proteins are often associated with cancer. Polycomb repressive complex 2 (PRC2) silences gene transcription by generating a lysine 27 trimethylation epigenetic marker on histone H3. Our laboratory is interested in the role of PRC2 in regulating malignant phenotypes of EOC.

Detailed descriptions

Molecular mechanism that reconciles the tumor-suppressing role of oncogene-induced senescence with the tumor-promoting nature of activated oncogenes

We discovered a novel cell-intrinsic mechanism by which activated oncogenes (such as RAS) promote senescence while at the same time predisposing cells to senescence bypass, by increasing the mutation rate and allowing for secondary mutagenic events.  We show that activation of oncogenes inactivates the BRCA1 DNA repair complex by dissociating BRCA1 from chromatin.  This event precedes senescence-associated cell cycle exit and coincides with the accumulation of DNA damage.  We have found that down-regulation of BRIP1, a physiological partner of BRCA1 in the DNA repair pathway, is sufficient to trigger BRCA1 chromatin dissociation.  Conversely, ectopic BRIP1 rescues BRCA1 chromatin dissociation and suppresses both oncogene-induced senescence and the DNA damage response.  In addition, we find that oncogene-induced upregulation of microRNA 29 suppresses the expression of B-Myb, leading to down-regulation of BRIP1.  Significantly, cells undergoing senescence do not exhibit a BRCA1-dependent DNA repair response when exposed to DNA damage, and DNA damage promotes senescence bypass and transformation.  In sum, our study provides a molecular basis by which activated oncogenes induce senescence, but at the same time increase mutation rate and allow for mutations that promote senescence bypass and ultimately drive cancer development.

Our future work will investigate the mechanism by which BRCA1 chromatin dissociation promotes senescence-associated cell cycle exit. In addition, we will determine the signaling pathway downstream of oncogenic-RAS that drives BRCA1 chromatin dissociation. Finally, we will examine the implication of this novel pathway in cancer and, in particular, in EOC.

In addition to increasing our understanding of the role of BRCA1 in cancer, the molecular insights gained from these studies will help to explain a major paradox in the senescence field: how activated oncogenes induce senescence but at the same time predispose cells to transformation.

Role of Wnt signaling in regulating senescence of EOC cells

We demonstrated that Wnt5a, a non-canonical Wnt ligand, is expressed at significantly lower levels in human EOCs compared with either normal ovarian surface epithelium or fallopian tube epithelium.  Notably, a lower level of Wnt5a expression correlates with tumor stage and predicts shorter overall survival in EOC patients. Promoter CpG island hypermethylation contributes to downregulation of Wnt5a observed in EOC cells.  Accordingly, restoration of Wnt5a expression inhibits the proliferation of human EOC cells both in vitro and in vivo in an orthotopic EOC mouse model.  Mechanistically, Wnt5a antagonizes canonical Wnt/b-catenin signaling and induces cellular senescence by activating the epigenetic histone repressor A (HIRA)/promyelocytic leukemia (PML) senescence pathway.   In summary, we showed that loss of Wnt5a predicts poor outcome in EOC patients and Wnt5a suppresses the growth of EOC cells by triggering cellular senescence.  Our data suggest that driving EOC cells to undergo senescence by reconstituting Wnt5a signaling represents a novel strategy for developing urgently needed EOC therapeutics.

We will investigate the effects of Foxy5, a Wnt5a derived hexapeptide that is sufficient to restore Wnt5a signaling, on malignant behavior of human EOC cells in vitro and in vivo in preclinical models. In addition, we will determine the role of Wnt5a down-regulation in oncogene-initiated transformation of ovarian surface epithelial cells and fallopian tube epithelial cells. Further, we will elucidate the molecular mechanism by which Wnt5a antagonizes canonical Wnt/b-catenin signaling in human EOC cells.

The molecular insights gained from these studies will not only contribute to the understanding of the mechanism whereby Wnt5a down-regulation contributes to EOC, but may eventually establish Wnt signaling as a bona-fide therapeutic target for EOC by driving EOC cells to undergo cell senescence.

Role of polycomb repressive complex 2 (PRC2) in EOC

Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of PRC2 that includes non-catalytic subunits SUZ12 and EED.  When present in PRC2, EZH2 catalyzes trimethylation on lysine 27 residue of histone H3 (H3K27Me3), resulting in epigenetic silencing of gene expression.  Here, we investigated the expression and function of PRC2 in EOC.  When compared to normal human ovarian surface epithelial cells or fallopian tube epithelial cells, EZH2, SUZ12 and EED were expressed at markedly higher levels in human EOCs.  Consistent with this premise, H3K27Me3 was also overexpressed in human EOC cells. Notably, EZH2 and SUZ12 expression positively correlated with each other and also correlated with expression of Ki67  (a marker of cell proliferation) in EOCs.  Inhibition of PRC2 by knocking down the expression of EZH2 or SUZ12 reduced the level of H3K27Me3 and suppressed the growth of human EOC cells both in vitro and in vivo in an orthotopic EOC model by triggering apoptosis of human EOC cells.  Finally, we showed that knockdown of EZH2 or SUZ12 suppressed the invasion of human EOC cells.  Together, these data support the premise that PRC2 promotes the proliferation and invasion of human EOC cells.  Recently we used a combination of whole genome ChIP-seq analysis and global gene expressing profiling to identify 60 EZH2 direct target genes in human EOC cells.  This list includes 56 novel putative EZH2 target genes in addition to 4 known EZH2 target genes.  Notably, we validated ALDH1A1, a putative marker of cancer stem cells of certain tumor types, as a direct EZH2 target gene.

Our future work will examine the function of the key EZH2 /PRC2 target genes in human EOC cells. In addition, we will elucidate the mechanism underlying EZH2 upregulation in human EOC cells. Further, we will investigate the effects of small molecule inhibitors of EZH2 on malignant behaviors of human EOC cells.

Together, these studies will provide molecular insights into how PRC2 contributes to the malignant behavior of human EOC cells. Ultimately, these studies have the potential to provide scientific rationale for targeting EZH2 as novel EOC therapeutics.