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Page 376 Sale et al. Cancer Drug Resist 2019;2:365-80 I http://dx.doi.org/10.20517/cdr.2019.14

influence this? Selumetinib withdrawal caused equivalent hyperactivation of ERK1/2 in both BRAF V600E - and
KRAS G13D -amplified/upregulated cells, suggesting that differences in the magnitude of ERK1/2 activation
were not responsible for the contrasting consequences of MEKi removal . One possible explanation is
[11]
that the activation of other KRAS effector pathways, or KRAS and/or ERK1/2-mediated processes such as
EMT, suppresses cell cycle arrest, senescence and/or cell death. Indeed, PI3K-PKB signalling downstream
of mutant RAS can attenuate RAS-induced senescence . Such a mechanism could explain why H6244-R,
[41]
which exhibit striking PI3K-PKB hyperactivation, did not undergo proliferative arrest despite very high
KRAS G13D expression, and strong ERK1/2 hyperactivation following MEKi withdrawal [Figure 5]. The
[11]
PI3K-PKB axis is also a well-recognised pro-proliferative and pro-survival pathway that could mitigate cell
cycle arrest or pro-death effects of excessive ERK1/2 activation in H6244-R cells [42,43] .

Alternatively the mutational or expression status of CDKIs and/or other tumour suppressors that comprise
the OIS circuitry could be an important factor. However, although mutations in key players such as p53
and CDKN2A (encodes p14ARF/p16INK4A) are present in these cell lines, their mutational and expression
status did not correlate with the phenotype of MEKi withdrawal . p53 expression was not increased by
[11]
selumetinib withdrawal (at least at 72 h) in any of the BRAF V600E - or KRAS G13D -amplified/upregulated cells,
and whereas COLO205 and HT29 cells harbour homozygous p53 mutations and did undergo cell cycle arrest
or death, HCT116 and LoVo express wild type p53 and proliferated normally upon withdrawal of MEKi .
[11]
Whilst the CDKIs p15 INK4B , p16 INK4A (mutated in HCT116), p19 INK4D and p21 may contribute to MEKi-
CIP1
withdrawal induced cell cycle aberrations in the BRAF V600E -amplified cells vs. the KRAS G13D -amplified/
upregulated cells in which no CDKI upregulation occurred, their induction by ERK1/2 activation was either
modest or expression levels extremely low; rather, cell cycle arrest following MEKi removal correlated with
and was wholly dependent on p57 KIP2 induction . It is unclear why p57 was only regulated in this manner
KIP2
[11]
in C6244-R, which underwent sustained p57 KIP2 -dependent cell cycle arrest upon ERK1/2 hyperactivation;
this mechanism was apparently uncoupled in HT6244-R or KRAS G13D -amplified/upregulated H6244-R and
L6244-R cells. CDKN1C (encoding p57 ) is known to be silenced by methylation in many tumour types
KIP2
so perhaps these HT6244-R, H6244-R and L6244-R cells exemplify this . Nevertheless, this upregulation
[44]
of p57 represents a novel tumour suppressive mechanism by which aberrant ERK1/2 signalling inhibits
KIP2
KIP2
proliferation and may promote senescence. Given that MEKi withdrawal increased CDKN1C/p57 mRNA
expression, ERK1/2 might activate transcription of CDKN1C/p57 in a manner analogous to regulation
KIP2
CIP1
of the closely related CDKI CDKN1A/p21 . Indeed, CDKN1C contains several classic ERK1/2-responsive
DNA-binding elements such as EGR1 and ETS [45,46] . Clearly, however, CDKN1C/p57 mRNA expression
KIP2
was not subject to the same stringent negative feedback that rapidly returned CDKN1A/p21 mRNA and
CIP1
protein to basal levels despite sustained ERK1/2 activation.
Whilst cell cycle arrest or cell death upon drug withdrawal was restricted to the MEKi-resistant cells with
BRAF V600E amplification, EMT was apparent only in KRAS G13D -amplified/upregulated cells despite similar
hyperactivation of ERK1/2 in all cases . MEKi withdrawal from BRAF V600E -amplified cells did not cause
[11]
repression of CDH1, or changes in other markers of EMT. Rather, these cells expressed significantly higher
levels of CDH1 than the KRAS G13D -amplified/upregulated cells regardless of the presence of MEKi . This
[11]
suggests that, on an epithelial-mesenchymal continuum, these BRAF V600E -amplified cells are more epithelial
in character, consistent with the parental cell lines having epithelial (BRAF V600E -mutant COLO205 and
HT29) or mesenchymal (KRAS G13D -mutant HCT116 and LoVo) EMT expression signatures . Whether this
[47]
reflects their distinct driving oncogenes, or reflects other genetic and/or epigenetic contexts that render
HCT116 and LoVo cells more mesenchymal and amenable to EMT upon ERK1/2 hyperactivation is unclear.
KRAS G13D amplification/upregulation, through the activation of other effector pathways, may provide the
required context for these cells to undergo ERK1/2-dependent EMT when MEKi is removed. PI3K-PKB
signalling, which is a known promoter of EMT and is upregulated in KRAS G13D -amplified H6244-R cells, is
an obvious candidate but was not required for repression of CDH1 upon MEKi withdrawal [11,48] .