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Aberrant ubiquitin-mediated proteolysis in oncogenesis

Final Report Summary - DGIRG (Aberrant ubiquitin-mediated proteolysis in oncogenesis)

A large body of experimental and clinical data indicates that defects in ubiquitin-dependent protein degradation are intimately linked with cancer pathogenesis. The main objective of our research is the elucidation of the molecular mechanisms by which deregulated function of SCF ubiquitin ligases contribute to oncogenesis. 

The specific aims of this project are:
Aim 1. To identify novel substrates for SCF ubiquitin ligases that have been shown to play roles in tumorigenesis.
Aim 2. To identify ubiquitin ligases for tumor suppressor proteins and proto-oncoproteins that are targeted by the ubiquitin-proteasome pathway.
Aim 3. To determine the contribution of defective SCF-mediated degradation to cancer.

Aim 1. To identify novel substrates for SCF ubiquitin ligases that have been shown to play roles in tumorigenesis.
To systematically identify novel cancer related substrates of ubiquitin ligases, we have developed and optimized several innovative immunopurification strategies followed by mass spectrometry analysis. We have identified seven novel cancer-related substrates of the SCFbetaTrCP ubiquitin ligase. We have focused on the degradation of one of them, namely, the Rap guanine exchange factor RAPGEF2. We found that in response to factors that induce cell migration, RAPGEF2 is rapidly phosphorylated by I-kappa-B-kinase-beta and casein kinase-1alpha and consequently degraded by the proteasome via the SCFbetaTrCP ubiquitin ligase. Failure to degrade RAPGEF2 in epithelial cells results in sustained activity of Rap1 and inhibition of cell migration induced by HGF, a potent metastatic factor. Furthermore, expression of a degradation-resistant RAPGEF2 mutant greatly suppresses dissemination and metastasis of human breast cancer cells. These findings reveal a molecular mechanism regulating migration and invasion of epithelial cells and establish a key direct link between IKKbeta and cell motility controlled by Rap-integrin signaling.

Aim 2. To identify ubiquitin ligases for tumor suppressor proteins and proto-oncoproteins that are targeted by the ubiquitin-proteasome pathway.
We have found that E2F3, a key regulator of cell cycle progression, becomes unstable as cells exit the cell cycle. E2F3 degradation is mediated by the anaphase-promoting complex/cyclosome and its activator Cdh1 (APC/CCdh1). E2F3 interacts with Cdh1, but not Cdc20, the other APC/C activator. Enforced expression of Cdh1 results in proteasome-dependent degradation of E2F3, whereas the over-expression of Cdc20 has no effect on E2F3 turnover. Finally, silencing of Cdh1 by RNA interference stabilizes E2F3 in differentiating neuroblastoma cells. These findings indicate that the APC/CCdh1 ubiquitin ligase targets E2F3 for proteasome-dependent degradation during cell cycle exit and neuronal differentiation.

Aim 3. To determine the contribution of defective SCF-mediated degradation to cancer.
We have generated knockin mice that conditionally express stabilizing mutations of REST, a master repressor of neuronal gene expression that is targeted for ubiquitin-mediated degradation by the SCFbetaTrCP ubiquitin ligase. This in vivo model is crucial since work focused on REST degradation has been performed in cultured cells with no in vivo analysis, which is essential for studying the contribution of defective REST degradation in cancer development. This animal model allows the expression of the degradation-resistant REST mutant in an inducible and tissue-specific manner. It represents a precious tool to examine whether defects in REST degradation, due to mutations in its phosphodegron, can affect malignant transformation in mice. As far as we know, this is the first animal model that allows the analysis of REST defective degradation in a spatially, e.g. in a specific tissue, and temporally, e.g. during embryonic development or cancer progression, regulated fashion.