Simvastatin Induced Translocation of Mutant KRAS in Colorectal Cancer Cells
L.L. Krens, C.M.C. Pullen, R.F. Baak-Pablo, H.J. Guchelaar, R.J.H.M. van der Straaten
Department of Clinical Pharmacy and Toxicology, Leiden University Medical Center, Leiden, The Netherlands
Introduction
Conclusions
Results
KRAS mutation status in colorectal cancer is
leading for the use of EGFR antibodies, such
as cetuximab. KRAS is activated by posttranslational modification (prenylation) by
binding C15 and C17 fatty acid chains. As a
consequence KRAS becomes more lipophilic
and associates with the membrane to exert its
function. Statins inhibit the synthesis of these
C15 and C17 chains. Statins may therefore
have the potential to influence the KRAS
mutant phenotype. Previous experiments have
shown that KRAS mutant cells could be
sensitized to anti-EGFR therapy using
simvastatin
This study sought to determine the mechanism
of sensitisation by simvastatin in KRAS mutant
CRC cells. We hypothesise that by inhibiting
the prenylation, KRAS will not associate with
the inner-membrane and KRAS will be less
active, making KRAS mutant cells more
susceptible to anti-EGFR therapy
§  KRAS is localized both in the cytoplasm
and in the plasma membrane of KRAS wild
type cells, the localization, however, in the
plasma membrane was much more
abundant in KRAS mutant cells.
In SW48, KRAS is localized in plasma membrane and also distributed through cytoplasm. Simvastatin does not affect the KRAS localization. KRAS expression is
abundant in plasma membrane of KRAS mutant cell lines SW480 and LoVo. It is evident that KRAS detaches from plasma membrane and translocates to the
cytoplasm when treated with simvastatin. KRAS expression in HCT116 is comparable with SW48 and no effect of simvastatin on KRAS localization was observed.
SW48
§  For KRAS wild type cells, no effect of
simvastatin on the amount of KRAS in
plasma membrane was observed
§  For KRAS mutant cells SW480 and LoVo,
a clear effect of simvastatin on
translocation of KRAS from plasma
membrane to the cytoplasm was observed.
Nevertheless, this effect was not observed
in HCT116.
Method
Discussion
LoVo (KRAS G13D), HCT116 (KRAS G13D),
SW480 (KRAS G12V) and SW48 (KRAS
wildtype) CRC cells were seeded in NuncTM
glass 8-chamber slides at density of 20,000
cells/well and cultured for 24 hours. After 24
hours, medium was replaced with medium with
or without simvastatin (2µM) and cultured for
another 24 hours. Cells were fixated with 2%
PFA, permeabilized using 0.1% Saponin + 2%
PFA, incubated with KRAS mouse anti-human
antibody and incubated with Alexa 488 goat
anti-mouse antibody. Vectashield-Dapi was
used to stain the nucleus.
Previous studies showed that activating
mutations in KRAS can predict resistance to
EGFR-targeted therapy.2 Circumventing this
activation by preventing the production of
farnesyl groups, may render these cells EGFR
dependent and consequently susceptible for
anti-EGFR therapy. This study shows that
mutant KRAS is abundant in the plasma
membrane of CRC cells LoVo and SW480, but
not in HCT116. Incubation with simvastatin
induces translocation of KRAS from plasma
membrane to cytoplasm in LoVo and SW480.
Whether this is due to decreased amount of
farnesyl groups, has not been tested.
However, because no translocation in HCT116
cells was observed, other mechanisms could
be involved in the detachment of KRAS by
simvastatin. For instance the effect of statins
on bone morphogenetic protein 1 , PKC
activation caused by statins3, or PI3K/AKT
downregulation4.
[email protected]
LoVo
SW480
Simvastatin
HCT116
+
Figure 1: Confocal pictures of cells incubated with or without simvastatin and stained for KRAS. Experiments are performed three times and representative pictures are
depicted. Upper panel show control cells (no simvastatin) and lower panel show cells incubated with simvastatin.
References:
1: Kodach L.L. et al. Gastroenterology 113, 1272-1281 (2007).
2: Van Houdt W.J. et al. Neoplasia 12, 443-452 (2010).
3: Manson J.C. et al. Circ. Res. 91, 696-703 (2002).
4: Bivona T.G. et al. Mol. Cell 21, 481-493 (2006).
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