Page 44 - Read Online

Page 154 Lotz et al. Cancer Drug Resist 2020;3:149-60 I http://dx.doi.org/10.20517/cdr.2019.114

ACETYLATIONS
We identified acetylation sites almost exclusively in the structured catalytic core of Top2a and only one
[16]
in the CTD after overexpression in yeast and BHK21 cells . This is consistent with the observation that
[38]
acetylations are often found in structured regions of proteins . In cancer cells, acetylations were mostly
identified in the CTD [39-41] [Figure 2]. Acetylations have not been studied as extensively as phosphorylations,
which limits the conclusions that can be drawn from a comparison between normal homeostasis and
cancer cells.

In healthy cells, the acetylations found in the DNA gate are located in the TOPRIM, coiled-coil, and C-gate
domains. Those located close to the extremities of the DNA groove could affect DNA binding [Figure 3A].
Interestingly, 8 acetylated positions in healthy cells located in the DNA gate have also been found
ubiquitinylated in cancer cell lines, suggesting a switch of modification in cancer cells [42-46] . Lys1196 was
found to be a site for acetylation in Jurkat cells, ubiquitination in Jurkat and HEP-2 cells, and SUMOylation
in HeLa cells [43,46,47] . This residue is located in a hinge region between the C-gate domain and the CTD and
could be involved in the regulation of Top2 activities [Figure 3B]. Lys278 in a loop region of the transducer
is acetylated in normal cells but found to be SUMOylated and ubiquitinylated in cancer cells [Figure 2].
Lys397, which lies in the transducer helix, is acetylated in cancer cells but can also be ubiquitinated.
These positions are mostly located on the surface of the N-gate domain and therefore compatible with the
addition of bulky modifications [Figure 3A]. Although buried at the dimeric interface, we also found an
acetylation on Lys168 in Top2a produced in the yeast expression system. Mutation of this residue showed
[16]
that this position is important for the coupling of ATPase activity and dimerization .
While few studies are available on the acetylation of Top2a in response to drugs, indirect inferences can
be made. The association of Top2 with histone deacetylases (HDAC) was shown to modulate its activity
and in particular etoposide-stimulated cleavage both in vivo and in vitro [48,49] . In addition, treatment of
[50]
hepatocellular carcinoma cells with an HDAC inhibitor triggers the proteasomal degradation of Top2 .

Interestingly, acetylations that were identified in BHK21 were also found as targets for ubiquitination
in cancer cells. Inversely, most acetylations identified thus far in cancer cells do not correspond to
reported ubiquitination or SUMOylation sites, which indicates that regulation of some PTM depends
on the cell state. Although further analysis is needed, these studies suggest a regulation of Top2a activity
by acetylations and a potential interplay between other modifications such as ubiquitinations and
SUMOylations.

UBIQUITINATIONS AND SUMOYLATIONS
Ubiquitin is a small regulatory protein that when attached to proteins alter their cellular localization,
protein activity, or molecular interactions, and may target them for proteasome degradation .
[51]
Ubiquitinations were identified throughout the Top2a sequence in cancer cells [Figure 2]. In non-cancer
cells (mouse embryonic fibroblasts), only one ubiquitination in the ATPase transducer domain has been
reported thus far. Lys378 is ubiquitinated by the E3 ligase activity of the APC/C complex, promoting 26S

proteasome degradation at G1 phase, thus modulating Top2a levels for chromosome maintenance . This
[52]
residue interacts directly with the ATP molecule and could be accessible to modifications when the N-gate
is open during the catalytic cycle.

Introduction or removal of ubiquitin is also a mechanism of drug resistance, as it modulates the Top2a
activities and protein levels through proteasome degradation [53-55] . Deficiency in the RNF168 E3 ubiquitin
ligase in breast cancer cells, or elevated levels of ubiquitin ligase Mdm2 in osteosarcoma cells, confers
resistance to the Top2 poison etoposide, by regulating Top2 activities [53,56] . Ubiquitin-mediated degradation