Keynote Conference - Interevent

Symp#14 Cell cycle control mechanisms
Chairs Patricia Gama and Hugo Aguirre Armelin
Stabilizing nuclear p27 kip1 with Skp2/Cks1 E3 ligase inhibitors as a potential therapeutic intervention for endometrial cancer and
other cancers
Savvas C. Pavlides, Lily Wu, Kuang-Tzu Huang, Stepahnie V. Blank, Khushbakhat Mittal, Timothy Cardozo, and Leslie I. Gold
School of Medicine, New York University, USA
The cell cycle is precisely regulated via the ubiquitin-proteasome system (UPS) by three substrate-specific E3 ubiquitin ligases, APC-
Cdc20, APC-Cdh1, and SCF-Skp2/Cks1. Inhibitors of specific E3 ligases that degrade proteins involved in cell cycle arrest are significant
targets to block for cancer therapy and a significant improvement over currently used non-specific proteasome inhibitors. The cyclindependent
kinase inhibitor, p27 kip1 (p27), is important for cell cycle arrest in G1. SCF-Skp2/Cks1 ubiquitylates nuclear p27 targeting it
for degradation thereby causing cell cycle progression. In turn, APC-Cdh1 signals Skp2 and Cks1 degradation, maintaining abundant
levels of p27 for cell cycle arrest. We show perpetual degradation of p27 by the UPS in type I endometrial carcinoma (ECA), an
estrogen (E2)-induced cancer. Using normal human primary endometrial epithelial cells (EECs), we demonstrate that E2 induces
MAPK-Erk2-driven ubiquitin-mediated degradation of p27 by its phosphorylation at T187, required for its degradation by SCF-
Skp2/Cks1. Also, E2 decreases APC-Cdh1 to increase Skp2 and Cks1 levels for p27 degradation. We propose that E2-induced
degradation is involved in the pathogenesis of ECA because knocking-down Skp2 completely blocks E2-induced p27 degradation and
growth stimulation. Conversely, progesterone (Pg) and TGF-β, both inhibitors of EEC growth, markedly increase p27 by increasing
Cdh1 thereby causing destruction of Skp2/Cks1 leaving p27 intact. Accordingly, separately knocking-down Cdh1 and TGF-β obviate
both Pg- and TGF-β-induced stabilization of nuclear p27 and completely blocks their ability to inhibit growth. These studies suggest
that preventing p27 degradation is a rational therapeutic strategy for the treatment of type I ECA. Indeed, small molecule inhibitors
of Skp2 E3ligase activity (Skp2E3LIs) shown in silico to block p27 binding to Skp2-Cks1, inhibit both E2-induced proliferation and
degradation of p27 in an ECA cell line and in primary ECA cells. Significant progress has been made in vitro showing lack of toxicity
and that certain Skp2E3LIs specifically block nuclear degradation of p27 where it can inhibit Cdk1 for cell cycle arrest. Skp2E3LIs
provide tools for understanding the role of the UPS in p27-mediated cell cycle regulation and as a novel approach to treating ECA and
many other cancers with inverse levels between p27 and Skp2.
Cyclin F-mediated degradation of RRM2 (Ribonucleotide Reductase family member 2) controls genome integrity and DNA repair
Vincenzo D’Angiolella 1 , Valerio Donato 1 , Frances M. Forrester 1 , Yeon-Tae Jeong 1 , Claudia Pellacani 1 , Yasusei Kudo 1,2 , Anita Saraf 3 ,
Laurence Florens 3 , Michael P. Washburn 3,4 , and Michele Pagano 1,5
1 Department of Pathology, NYU Cancer Institute, New York University School of Medicine, 522 First Avenue, SRB 1107, New York, NY
10016, USA. 2 Department of Oral Molecular Pathology, Institute of Health Biosciences, The University of Tokushima Graduate School,
Tokushima, Japan. 3 The Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, MO 64110, USA. 4 Department of
Pathology and Laboratory Medicine, The University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, Kansas 66160,
USA. 5 Howard Hughes Medical Institute
F-box proteins are the substrate recognition subunits of SCF ubiquitin ligase complexes. The F-box protein family obtained its name
from Cyclin F (also known as Fbxo1), in which the F-box motif was first described. Cyclin F is localized both to the centrosomes and
the nucleus. At the centrosomes, Cyclin F targets CP110 for proteasomal degradation during G2 to limit centrosome duplication to
once per cell cycle. Instead, the nuclear function of Cyclin F remains elusive. Using purifications and mass spectrometry, we
identified RRM2 (the ribonucleotide reductase family member 2) as a new interactor of the F-box protein Cyclin F. Ribonucleotide
reductase (RNR) catalyzes the conversion of ribonucleotides to the deoxyribonucleotides (dNTPs) that are necessary for replicative
and repair DNA synthesis. Because of this fundamental function, RNR is among the most well-conserved (from prokaryotes to
eukaryotes) and highly-regulated enzymes. Indeed, an unbalanced and/or increased dNTP pools produce a hypermutator phenotype,
and decreased dNTP levels interfere with proper DNA replication and repair. We found that, during G2, following CDK-mediated
phosphorylation of Thr33, RRM2 is degraded via SCF Cyclin F to maintain balanced dNTP pools and genome stability. After DNA damage,
Cyclin F is downregulated in an ATR-dependent manner to allow accumulation of RRM2. Defective elimination of Cyclin F delays DNA
repair and sensitizes cells to DNA damage, a phenotype that is reverted by expressing a non-degradable RRM2 mutant. In summary,
we have identified a novel biochemical pathway that controls the abundance of dNTPs and ensures efficient DNA repair in response
to genotoxic stress.
Resveratrol and Temozolomide co-treatment induces mitotic catatrophe and senescence in glioma cells through modulation of
mitotic regulators
Eduardo C. Filippi-Chiela and Guido Lenz*
Department of Biophysics and Center of Biotechnology,
Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.
Email: lenz@ufrgs.br Phone: 55 51 33087613
Blockage of the cell cycle is an important strategy in cancer therapeutics. On the other hand, forcing mitosis in cells with high levels of
DNA damage may also be a good strategy, since it may induce mitotic catastrophe (MC) and senescence. Temozolomide (TMZ), the
primary therapy used in gliomas, causes DNA damage and G2 arrest. Resveratrol (Rsv) presents additive toxicity with TMZ in several
glioma cells in vitro and in vivo, but the mechanism of additive toxicity is not clear, which is the aim of the present work. Rsv
abrogated the TMZ-induced G2 arrest when added together, but not after TMZ. Rsv potentiated the increase in TMZ-induced
gammaH2AX, but not ATM and Chk2. Abrogation of TMZ-induced cell cycle arrest by Rsv involved a reduction of cyclin D,
pWee1(S642) and of the Wee1 target site, pCdc2(Y15) and increase of cyclin B. This suggests a state of forced passage through G2
checkpoint despite large DNA damage, a scenario typical of MC. In order to quantify MC and senescence, we developed a quantitative
method called nuclear morphometric analysis (NMA). Indeed, after acute treatment with Rsv+TMZ, the proportion of cells with high
nuclear irregularity increased from 5 to 28% in 48h. Seven days later, a large induction of senescence and reduction in clonogenicity
was observed. In conclusion, presence of Rsv forced damaged cells treated with TMZ through mitosis due to a reduction of Wee1 and
pCdc2(Y15), leading to MC and senescence. Funding: CNPQ and FAPERGS. No conflict of interest.
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