ABSTRACTS – ORAL PRESENTATIONS - AMCA, spol. s r.o.

P34. PHARMACOLOGICAL INHIBITION OF SKP2-SCF COMPLEX ACTIVITY AS AN
APPROACH TO MODULATE CHARACTERISTICS OF CANCER STEM CELLS
Šárka Šimečková 1,2* , Zuzana Pernicová 1,3 , Radek Fedr 1 , Alois Kozubík 1,2 , Karel Souček 1,3*
1
Department of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech
Republic, Brno, Czech Republic;
2
Department of Experimental Biology, Faculty of Science, Masaryk University, Brno,
Czech Republic;
3
Center of Biomolecular and Cellular Engineering, International Clinical Research Center,
St. Anne´s University Hospital Brno, Brno, Czech Republic
*correspondence to: simeckova@ibp.cz, ksoucek@ibp.cz
The cancer stem cells (CSCs) represent an attractive target for anticancer therapy. These
cells are responsible for tumor relapse and serve as a reservoir of cancer cells within
tumors. There are some characteristics of CSCs, which they share with their normal
counterparts, as for example asymmetric division, differentiation into heterogeneous cell
populations, and slow cycling. These cells have also an ability to invade into a surrounding
tissue and importantly, they are more resistant to conventional drug treatment.
Skp2 protein is a crucial component of Skp2-SCF complex that functions as an E3
ubiquitin ligase involved in protein degradation. Skp2 is essential for cell cycle regulation
via degradation of p27 Kip1 , Cdt1 and other molecules. High level of Skp2 was described
in various types of cancer, including prostate and colorectal cancer. Pharmacological
inhibition of Skp2-SCF complex has strong potential in anticancer therapy. Binding NEDD8
to Cullins is necessary for Skp2-SCF formation and activity (Kawakami et al., 2001).
Recently, a novel inhibitor of NEDD8 pathway MLN-4924 was described (Soucy et al.,
2009). Disruption in NEDD8 pathway using this inhibitor leads to accumulation of Skp2
substrates and triggers cellular processes such as cell cycle arrest, cellular senescence
and apoptosis.
In this work we used MLN-4924 inhibitor and investigated its effect on murine
adenocarcinoma cell line cE2 and human colorectal carcinoma cell line HCT116.
Accumulation of Skp2-SCF substrates after MLN-4924 treatment led to deregulation of
cell cycle in both cell lines. Interestingly, both cell lines express surface markers CD133
and CD44 typical for CSCs and can be divided into two subpopulations based on the
expression of CD133 – CD44 + CD133 low and CD44 + CD133 high (Fedr et al., 2013). Therefore,
we further investigated the effect of MLN-4924 inhibitor on these subpopulations. MLN-
4924 reduced clonogenic capacity of single cells in both cell lines and downregulated
surface expression of CD133. Next, using flow cytometry we introduced a multicolor
multiparametric protocol for simultaneous assessment of expression of surface CSCs
markers, DNA synthesis, viability, cell cycle, apoptosis, and DNA damage. Using HCT116
cells, we show that CD44 + CD133 low and CD44 + CD133 high subpopulations significantly
differ in their response to treatment with MLN-4924 inhibitor. Higher sensitivity to DNA
damage induction and arrest in G1 phase of the cells cycle was found in CD44 + CD133 low
subpopulation, whereas CD44 + CD133 high subpopulation arrested preferentially in S phase
and DNA damage was not observed.
128 Analytical Cytometry VII