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Sale et al. Cancer Drug Resist 2019;2:365-80 I http://dx.doi.org/10.20517/cdr.2019.14 Page 377

Several reports have suggested that EMT can override OIS, and that ZEB1 often plays an important role [49-51] .
ZEB1 has been suggested to suppress p15 INK4B , p16 INK4A and p21 transcription to maintain cell proliferation [50,51] ,
CIP1
KIP2
though it is unclear whether ZEB1 and EMT can repress or regulate p57 . Thus in KRAS G13D -amplified/
upregulated H6244-R and L6244-R cells, which undergo a ZEB1-dependent EMT , progression to OIS
[11]
following MEKi withdrawal may be inhibited by EMT. EMT can also protect against apoptosis and cell
death [38,39,52] , which is consistent with the resistance to classic chemotherapeutics exhibited by H6244-R and
L6244-R following EMT. This raises the intriguing possibility that suppression of EMT when ERK1/2 are
hyperactivated following MEKi removal could render H6244-R and L6244-R vulnerable to ERK1/2-driven
senescence or cell death. This in turn raises the question of whether enforced EMT can protect BRAF V600E -
amplified C6244-R or HT6244-R from proliferative arrest, senescence or cell death upon MEKi withdrawal.
Thus ERK1/2 hyperactivation, in the context of KRAS G13D amplification/upregulation, could mitigate its own
tumour suppressive effects by triggering an EMT.

Finally, why KRAS G13D -mutant HCT116 and LoVo cells consistently adapt to MEKi by reinstating ERK1/2
phosphorylation and pathway output to precisely parental levels is unclear . Evidently BRAF V600E -mutant
[11]
COLO205 and HT29 cells must adapt by reimposing p-ERK1/2 within a narrow sweet-spot to avoid cell
cycle arrest, senescence or death: clones with lower or higher p-ERK1/2 in the presence of MEKi will be out-
competed by clones with parental p-ERK1/2. However, given that H6244-R and L6244-R grew normally when
ERK1/2 were hyperactivated, there is no obvious selection pressure to prevent the emergence of selumetinib-
resistant HCT116 and LoVo clones with higher than parental levels of p-ERK1/2 . This suggests that clones
[11]
with higher levels of KRAS G13D amplification/upregulation either do not arise at all, occur at some cost that is
not immediately apparent and are selected against or rheostat mechanisms in the pathway maintain ERK1/2
phosphorylation at this level regardless of higher order KRAS G13D amplification or expression.

CONCLUSION
Our results have defined p57 expression as a novel tumour suppressive mechanism that responds to
KIP2
inappropriately activated ERK1/2. Thus, p57 KIP2 joins p16 INK4A and p21 as ERK1/2-responsive CDKIs that
CIP1
mediate cell cycle arrest and/or senescence in response to high levels of ERK1/2 signalling. Our results also
KIP2
define p57 as a genetic link between high level ERK1/2 signalling and the reversibility of MEKi-resistance,
suggesting that a cell autonomous ERK1/2-p57 KIP2 pathway selects against those cells with BRAF V600E
amplification. Various cellular contexts probably contribute to the different phenotypes observed upon
MEKi-withdrawal; for example, in BRAF V600E -amplified HT6244-R cells the failure to upregulate p57 KIP2
and sustain a G1 arrest allows cells to progress instead to cell death, which also selects against BRAF V600E
amplification to reverse resistance. These results provide a molecular explanation, and a further rationale,
for drug holidays and intermittent dosing strategies as a means of mitigating or delaying acquired resistance
to ERK1/2 pathway inhibitors in cases of resistance driven by BRAF V600E amplification. However, caution
must be exercised in applying such strategies in the case KRAS G13D amplification, where MEKi withdrawal
promoted EMT, cell motility and chemoresistance, phenotypes that are highly undesirable for a drug holiday
regimen. Thus reversibility of MEKi-resistance and the consequences of MEKi withdrawal may be influenced
by the nature of the particular amplified oncogene - BRAF or KRAS - highlighting again the challenges of
targeting cancers with KRAS mutations.

DECLARATIONS
Acknowledgments
We thank all members of the Cook laboratory and our collaborators who contributed to the original study
upon which this article is based.

Authors’ contributions
All authors contributed to the conceptualisation and writing of this article.