p53 is a critical tumor suppressor, as evidenced by its frequent mutation in human cancers and the completely penetrant tumor predisposition of p53 null mice. p53 acts as a tumor suppressor by inducing cell cycle arrest or apoptosis in response to stress signals. To understand the basis for p53 driving different cellular responses, we previously conducted a screen to identify target genes of p53 activated selectively during apoptosis but not cell cycle arrest. Using gene expression profiling, we identified the pro-apoptotic gene Siva (CD27-binding protein), which encodes a protein shown previously to interact with death receptors. Our initial characterization of Siva showed that it is both necessar and sufficient for p53-dependent apoptosis in primary cerebellar granular neurons. Interestingly, in our studies of a p53 transcriptional activation mutant that is unable to efficiently activate th vast majority of known p53 target genes, yet is completely competent as a tumor suppressor, we found that Siva is one of the small set of tumor-suppression-associated p53 target genes induced by this transactivation mutant. These findings suggest Siva could be a critical mediator of p53-tumor suppression. In addition, our studies using Siva null mice we generated have revealed a critical role for Siva during early embryonic development. Siva-deficiency results in embryonic lethality associated with defects in extra-embryonic vasculature remodeling and neural tube closure, phenotypes that resemble those seen in TGF-beta pathway component knockout mice. Moreover, our characterization of Siva-null embryos revealed a deregulation of signaling in the SMAD5 pathway downstream of TGF-beta family receptors. These findings, combined with the observation of ours and others that Siva can interact with TGF-Beta Activating Kinase-1 (Tak1), a mediator of TGF-beta signaling, suggest a vital role for Siva in TGF-beta superfamily signaling. However, the importance of Siva's signaling role in the TGF-beta pathway for Siva apoptotic function is unclear. Moreover, Siva's role in the p53 cellular functions of apoptosis, G1 arrest and senescence and in p53- mediated tumor suppression in vivo remain incompletely understood. We will cross Siva conditional knockout mice (Sivafl/fl) that we generated with Rosa-26-Cre-ER mice to ubiquitously delete Siva through tamoxifen treatment and assess the effect of Siva-deficiency on apoptosis levels in radiosensitive tissues known to display p53-dependent apoptosis following ionizing radiation. These studies will reveal the contexts in which Siva is important for p53-mediated apoptosis. In addition, we will also evaluate the as yet unknown role of Siva in the p53 cellular functions of cell cycle arrest and senescence using mouse embryonic fibroblast model systems. Together, these approaches will provide a complete understanding of Siva's function downstream of p53. To investigate the unknown role of Siva in tumor suppression, we will cross the Siva conditional knockout mice to E?-myc transgenic mice, which are prone to B-cell lymphomagenesis and in which p53-mediated apoptosis is central for tumor suppression. Fetal liver hematopoetic stem cells (HSCs) will be derived, transduced with retroviral MSCV-Cre-ER-Luciferase, and injected into lethally irradiated recipient mice and lymphomagenesis will be monitored. These studies will reveal whether Siva has tumor suppressor activity. To elucidate the importance of the Siva-Tak1 interaction in Siva- and p53-mediated apoptosis, we will define domains of Siva critical for Tak1 interaction and evaluate the role of Siva's E3 ligase activity in ubiquitylating Tak1. We will then assess the requirement for the Siva-Tak1 interaction and Siva ubiquitylation activity for Siva-induced apoptosis in E1A;p53-/- MEFs. These studies will determine whether Siva's function in TGF-beta signaling is important for Siva-induced apoptosis and thus provide insight into the role of TGF-beta signaling in p53-dependent apoptosis. Together, these experiments will elucidate the role of Siva in p53-dependent effector functions and tumor suppression as well as providing insight into the significance of TGF-beta signaling in Siva- and p53-mediated apoptosis.

Public Health Relevance

Siva is a direct target of the critical tumor suppressor p53 during the process of cell death called apoptosis, yet Siva's role in apoptosis in vivo and in tumor suppression is unknown. In this project, we will clarify the requirement for Siva in apoptosis and tumor suppression as well as the biochemical mechanism of action of Siva. The results from these studies will increase the knowledge of how p53 exerts its tumor suppressive effects, and may aid in the design and development of better cancer diagnostics and therapies.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
5F31CA167917-02
Application #
8458195
Study Section
Special Emphasis Panel (ZRG1-F09-D (08))
Program Officer
Schmidt, Michael K
Project Start
2012-03-15
Project End
2017-03-15
Budget Start
2013-03-15
Budget End
2014-03-15
Support Year
2
Fiscal Year
2013
Total Cost
$42,232
Indirect Cost
Name
Stanford University
Department
Pediatrics
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Van Nostrand, Jeanine L; Brady, Colleen A; Jung, Heiyoun et al. (2014) Inappropriate p53 activation during development induces features of CHARGE syndrome. Nature 514:228-32