Defective cell cycle regulation leads to genomic instability and ultimately cancer development. The Anaphase Promoting Complex (APC) regulates cell cycle progression by forming two distinct sub-complexes, APCCdh1 and APCCdc20, to promote the timely ubiquitination and degradation of various cell cycle regulators. The APC activator Cdh1 has been implicated in tumorigenesis, while its exact roles in tumor suppression remain largely unclear. Therefore, the primary objective of this proposal will be to continue our previous efforts in examining both in vitro and in vivo how Cdh1 functions to block cancer development and progression by its interplay with multiple major tumor suppressor and oncogenic pathways. We previously published that APC- free Cdh1 could augment the catalytic activity of HECT domain-containing E3 ligase Smurf1 to regulate osteoblast differentiation. Furthermore, we obtained preliminary results showing that in addition to Smurf1, Cdh1 could also modulate the E3 ligase activity of WWP2, another NEDD4 family member, in an APC- independent manner. More importantly, we showed that depletion of Cdh1 enhanced WWP2-mediated degradation of its ubiquitin substrate, PTEN.
In Aim #1, we propose to use multiple genetic and biochemical approaches to identify the novel APC-independent molecular mechanism(s) by which Cdh1 controls WWP2 E3 ligase activity to govern timely degradation of PTEN, and to evaluate whether elevation of Akt signaling has a critical role in loss of Cdh1-mediated cancer development in vivo.
In Aim #2, we will characterize a multi- mechanistic role for Cdh1 in inhibiting tumorigenesis by suppressing BRaf activity. Our preliminary data reveal Cdh1 as a novel negative regulator of the BRaf/ERK oncogenic signaling through multiple mechanisms in a context-dependent manner. In primary cells, APCCdh1 earmarks BRaf for ubiquitination-mediated destruction, while in cancer cells, Cdh1 inhibits BRaf kinase activity largely by disrupting BRaf dimers in an APC- independent manner. In this Specific Aim, we will further characterize the molecular mechanism by which APCCdh1 targets BRaf for ubiquitination-mediated degradation. We will also examine how APC-free Cdh1 may inhibit BRaf dimerization leading to decreased ERK activity to influence tumorigenesis. Furthermore, we will also identify a possible dual regulatory mechanism in BRafV600E melanoma cells where both elevated activity of ERK and Cdk4/CyclinD1 may inhibit APCCdh1 by directly phosphorylating Cdh1. More importantly, using multiple in vitro cell culture and in vivo engineered animal models, we will further determine the significance of both APC-dependent and -independent regulatory pathways in mediating the tumor suppressive capabilities of Cdh1. Altogether, the proposed studies will not only provide novel mechanistic insights into the role of Cdh1 in tumorigenesis, but will also offer the rationale and molecular basis for the development of targeted therapeutics to inhibit downstream targets of Cdh1 such as WWP2 in modulating PTEN/Akt signaling (Aim #1) in breast cancer setting, and BRaf/ERK signaling (Aim #2) that plays driving roles in melanoma development.

Public Health Relevance

APC/Cdh1, whose function has been implicated in DNA damage repair and tumor suppression, is a critical regulator that governs cell cycle progression. However, the underlying molecular mechanisms are unknown. We plan to study the interplay between Cdh1 and the PTEN/Akt and BRaf/ERK signaling pathways, thus uncovering the regulatory functions of Cdh1 in tumor suppression. These studies will provide valuable insight into how Cdh1 integrates into the network of tumor suppressor/oncogene pathways to suppress tumor development, and will also provide the rationale and molecular basis for developing WWP2 inhibitors, or synergistic usage of Cdk4 inhibitors with PLX4302 as novel anti-cancer therapies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM089763-06
Application #
8957724
Study Section
Molecular Oncogenesis Study Section (MONC)
Program Officer
Hamlet, Michelle R
Project Start
2009-08-06
Project End
2019-06-30
Budget Start
2015-08-05
Budget End
2016-06-30
Support Year
6
Fiscal Year
2015
Total Cost
$334,950
Indirect Cost
$142,450
Name
Beth Israel Deaconess Medical Center
Department
Type
DUNS #
071723621
City
Boston
State
MA
Country
United States
Zip Code
02215
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