Projects and Grants
Project 1: Effectors of p53 and Pro-Survival and Pro-Apoptotic Signaling Pathways
Stuart A. Aaronson, MD, Program Director, Leader Project 1
Sam Lee, PhD, Co-Leader Project 1
p53 is widely known as the “guardian of the genome” due to its ability to activate either cell cycle arrest or apoptosis in response to DNA damage. This project has uncovered a novel role of p53 cell stress responses in enforcing innate immunity by transcriptional upregulation of IRF9, a central component of the type I interferon (IFN) response.
Recent evidence from our studies and others indicates that p53 also directly upregulates several target genes in pathways that play a major role in innate immunity, including toll-like receptors (TLRs), IFN-regulatory factors (IRFs), IFN-stimulated genes (ISGs), and tumor necrosis factor alpha (TNF-α). Within this project, we discovered a novel p53 target gene, CDIP (Cell Death Involved p53 target), which markedly upregulates p53-dependent expression of TNF-α and promotes TNF-α apoptosis over survival cell fate decisions.
Our central hypothesis is that p53 mediates pro-survival signaling involved in antiviral and inflammatory responses. These pathways also affect the tumor microenvironment to influence tumor progression.
Our specific aims are:
- To investigate the contribution of p53-dependent expression of these newly identified target genes to the innate antiviral immune response, including an miRNA component of this response identified by us to play a role as well. With Projects 2 and 3 we will explore our findings that type I IFNs enhance protein levels by a non-transcriptional mechanism as well as the post-translational modifications involved. We will also determine the ability of MDM2 inhibitors, which increase p53 at the protein level, to enhance p53-dependent innate antiviral responses in vitro and in vivo.
- To investigate mechanisms by which CDIP enforces TNF-α-induced apoptosis over survival and its specific role in TNF-α growth inhibition of human tumor cells. We will utilize a tandem affinity purification strategy to identify CDIP-interacting proteins and collaborate with Project 2 to solve the novel structure of this molecule. We have evidence for the feasibility of generating a CDIP knockout mouse that should help to elucidate CDIP tissue expression and any developmental perturbations due to loss of function on development, as well as how loss of CDIP function affects chemo/irradiation and TNF-α sensitivity.
- To integrate these investigations toward elucidating how p53 innate immune functions through cytokine signaling impact tumorigenesis in chemical carcinogenesis models in which endogenous IFNs and TNF-α are known to inhibit tumor formation. MDM2 small-molecule antagonists will be applied to dissect p53 tumor suppressor functions in tumor initiation and progression. These investigations will be aided by continuing monthly meetings and collaborative interactions involving experienced investigators within the program, each of whom is focused on studying novel aspects of p53 regulation and effectors.
Icahn School of Medicine, New York, NY (MSSM)
Massachusetts General Hospital (MGH)
Project 2: Molecular Interactions and Regulation of p53
Ming-Ming Zhou, PhD, Leader Project 2
James Manfredi, PhD, Co-Leader Project 2, Director Core B
Site-specific post-translational modifications of human tumor suppressor p53 induced by stress play an important role in the activity of p53 as a transcription factor that regulates cell cycle arrest, senescence, or apoptosis. Our long-term goal is to achieve mechanistic understanding of the molecular interactions and regulation of p53 in human biology and cancer.
While multiple acetylation and methylation sites in p53 have been reported, specific effects of individual or combined modifications on p53 activity remain elusive. Preliminary data are presented involving a structure-based functional analysis of p53, supporting the notion that acetylation-induced p53 activation in response to DNA damage is involved in co-activator recruitment and subsequent histone acetylation required for target gene transcriptional activation.
Our study specifically supports the notion that p53 recruitment of the co-activator CBP (CREB binding protein) requires association of the CBP bromodomain with p53 at acetylated lys382. This molecular interaction is essential for p53-induced transcriptional activation of the cyclin-dependent kinase inhibitor p21, which is involved in G1 cell cycle arrest.
Our central hypothesis is, therefore, that distinct modifications of p53, including lysine acetylation and methylation, have differential effects on p53 functions in cells. We propose a multifaceted approach to address mechanistic underpinnings of p53 transcriptional activation with an emphasis on the role of post-translational modifications in p53 activation.
Our specific aims are:
- To elucidate the molecular basis of these modification-mediated molecular interactions of p53 with co-activators and to develop small-molecule chemical probes with structure-based design to functionally modulate p53 interactions.
- To determine the interplay between the co-activators CBP/p300 and p53 C-terminal domain in transcriptional regulation and tumor suppression of p53 using a variety of biochemical and cell biological approaches, including the establishment of an in vivo model.
We expect our studies to yield new mechanistic understanding of post-translational modifications in p53 function. Given the central role of p53 in cancer, these studies will have important implications for the prognosis and treatment of human tumors.
Icahn School of Medicine, New York, NY (MSSM)
Project 3: Anti-Repression Mechanisms in p53-Mediated Stress Responses
Wei Gu, PhD, Leader Project 3
In the past 10 years, our lab has made significant contributions to understanding the dynamic pathway of p53 regulation by protein modifications. p53 functions as a central node for determining whether the cell responds to various types and levels of stress via apoptosis, cell cycle arrest, senescence, DNA repair, cell metabolism, autophagy, or aging. While the exact molecular events for p53-mediated choice of cell fate are still insufficiently explained, p53-controlled transactivation of target genes constitutes an essential event in each stress response pathway.
As a transcription factor, p53 demands an exquisitely complicated network of control and fine-tuning mechanisms to ensure correct, differentiated responses to the various stress signals encountered by cells. p53 was the first non-histone protein known to be regulated by acetylation (Gu and Roeder, 1997) . The acetylation levels of p53 are significantly enhanced in response to stress, and these levels correlate well with p53 activation and stabilization in all cell types in response to almost every type of stress.
Following our early findings of C-terminus p53 acetylation, we and others recently showed that p53 is also acetylated by Tip60/hMOF at residue K120 within the DNA binding domain (Tang et al., 2006; Sykes et al., 2006). K120 acetylation is crucial for p53-mediated apoptosis but has no obvious effect on p21 expression, an essential target of p53-mediated growth arrest.
More recently, we have identified K164 as a new site for in vivo acetylation of p53 by CBP/p300 and evaluated its function in p21 activation. Although acetylation defects at each individual site (K164, K120, and C-terminus) can be compensated by the modification of other sites, loss of acetylation at all these major sites completely abolishes p53’s ability to activate its mediated cell growth arrest and apoptosis.
These studies demonstrate an indispensible role of acetylation in p53 regulation (Tang et al., 2008). In contrast, p53 is tightly regulated by Mdm2 and its related protein Mdmx. Nonetheless, the molecular mechanisms by which p53 activity is controlled by Mdm2 and Mdmx are complex (Brooks and Gu, 2006). Mdm2 and MdmX are structurally related proteins, and physiological levels of both proteins are required in a non-redundant manner to balance p53 activity during embryonic development.
Notably, our recent studies show that acetylation of p53 abrogates Mdm2- and Mdmx-mediated repression by blocking the recruitment of Mdm2 or Mdmx to p53-responsive promoters (Tang et al., 2008). Our study identifies anti-repression as a major mechanism for acetylation-mediated p53 activation in stress responses.
Our central hypothesis is that protein modification of p53, such as acetylation, plays a major part in the scope of controlling p53 function by directly affecting the interactions of p53 with Mdm2/Mdmx or other cellular regulators in vivo, which enables p53 to activate transcription in a promoter-specific manner.
Our specific aims are:
- To elucidate the precise roles of p53 acetylation in releasing the repression of p53 from Mdm2 and Mdmx.
- To identify additional cellular factors involved in anti-repression mechanisms and investigate the roles of these factors in modulating p53 activities in stress responses.
Columbia University, New York, NY (MSSM)