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Page 366 Sale et al. Cancer Drug Resist 2019;2:365-80 I http://dx.doi.org/10.20517/cdr.2019.14

Keywords: BRAF, CDKN1C/p57 KIP2 , EMT, ERK, KRAS, MEK, MEK inhibitor, resistance, selumetinib

INTRODUCTION
The RAS-RAF-MEK1/2-ERK1/2 signalling pathway is deregulated in a variety of cancers due to mutations
in pathway components, most notably BRAF and the RAS isoforms. Consequently this pathway has been the
focus of major drug discovery efforts and numerous small molecule inhibitors of RAF, MEK1/2 or ERK1/2
kinase activities have been developed. Several of these have proven successful in the clinic, including the
MEK1/2 inhibitors (MEKi) trametinib and cobimetinib, and the BRAF inhibitors (BRAFi) vemurafenib and
dabrafenib, all of which are approved for the treatment of BRAF V600E/K -mutant melanoma . Various other
[1,2]
MEKi are in later stage clinical trials, including selumetinib (AZD6244/ARRY-142886) which is in phase III
clinical trials . MEKi are exquisitely selective because they bind within an allosteric pocket adjacent to
[2-5]
the catalytic site that is unique to MEK1 and MEK2. MEKi also inhibit ERK1/2 signalling in RAS-mutant
or wild type cells, whereas BRAFi actually promote pathway activation in these contexts and only inhibit
ERK1/2 in BRAF-mutant cells . Therefore MEKi have broader utility, but a narrower therapeutic margin,
[1,6]
than BRAFi.

As with all current targeted cancer therapeutics, MEKi efficacy is limited by innate and acquired resistance
and we have contributed to the understanding of both modes of MEKi resistance in colorectal cancer (CRC)
cells, where BRAF and KRAS mutations are common oncogenic drivers. For example, innate resistance to
MEKi is driven by strong PI3K-PKB signalling . CRC cells with BRAF or KRAS mutations evolve resistance
[7]
to MEK1/2 inhibitors by amplifying their mutant BRAF or KRAS alleles, or through emergent mutations in
MEK1 [8-11] . Amplification of the driving BRAF or KRAS oncogene results in overexpression of the respective
oncoprotein, which in turn causes hyperphosphorylation and activation of MEK1/2. This enlarged pool of
active MEK1/2, although restrained by the presence of MEKi, is sufficient to reinstate ERK1/2 phosphorylation
and activation to overcome these inhibitors. Indeed, the levels of ERK1/2 phosphorylation and pathway
output are reinstated to precisely that seen in parental, drug-naïve levels. Thus CRC cells evolve resistance
to MEKi through profound upstream pathway activation that sufficiently overcomes the presence of MEKi
to maintain ERK1/2 activity and drive proliferation and survival. A consequence of this mechanism of
resistance is that in the absence of MEKi the large pool of p-MEK1/2 is no longer restrained and so MEKi
withdrawal promotes rapid and sustained ERK1/2 hyperphosphorylation [9,11] .

Whilst moderate ERK1/2 activity is a well-established pro-proliferative and pro-survival signal [12,13] ,
excessive ERK1/2 signalling can trigger tumour suppressive mechanisms that ultimately lead to cell cycle
arrest, senescence and/or cell death [12,14] . Cell cycle arrest in response to high RAF activity has been shown
to be dependent on the cyclin-dependent kinase inhibitor (CDKI) p21 CIP1[15,16] ; indeed, ERK1/2 can promote
CDKN1A (encodes p21 ) transcription by activating ETS and C/EBP transcription factors and promoting
CIP1
their binding to multiple elements within a CDKN1A enhancer . Oncogenic RAS and RAF can also promote
[17]
irreversible cell cycle arrest or oncogene-induced senescence (OIS) that has been shown to be dependent
on ERK1/2 signalling, as well as p38 activity [18-20] . RAS-induced OIS is typically associated with, and often
dependent upon, upregulation of p14 , p16 INK4A , p21 and/or p53 [18,21-23] .
ARF
CIP1
ERK1/2 hyperactivation can also initiate or contribute to apoptotic cell death in some contexts . Mechanisms
[14]
include upregulation of death receptor ligands, such as TNF and FASL, or the death receptors themselves,
including FAS, DR4 and DR5, which promote the extrinsic pathway of apoptosis [24-28] .

In this commentary we discuss results from our recent study , including a novel tumour suppressive
[11]
pathway activated by excessive ERK1/2 signalling involving expression of the CDKI p57 KIP2 , encoded by
CDKN1C. p57 KIP2 expression is strongly linked to the magnitude of ERK1/2 signalling and drives cell cycle