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Page 372 Sale et al. Cancer Drug Resist 2019;2:365-80 I http://dx.doi.org/10.20517/cdr.2019.14

cells, KRAS G13D amplification reinstated ERK1/2 activity and pathway output to parental levels in selumetinib
resistant HCT116 (H6244-R) cells and these cells exhibited strong ERK1/2 hyperactivation following
MEKi withdrawal [9,11] . HCT116 cells also harbour an H1047R mutation in the PI3K catalytic subunit p110α
(encoded by PIK3CA), and unlike the BRAF V600E -amplified cells, KRAS G13D amplification also activated
PI3K-PKB signalling regardless of whether selumetinib was present [Figure 4] [9,11] . Remarkably, KRAS G13D
amplification and resistance to selumetinib were not reversible, even when drug was withdrawn for long
periods (> 6 months) [Figure 5] . In the shorter-term, these cells did not exhibit a proliferative defect, any
[11]
alteration in cell cycle profile, any upregulation of CDKIs or cell death when deprived of selumetinib, and
grew normally in vivo. In another KRAS-mutant CRC cell line, LoVo, acquired resistance to selumetinib was
associated with upregulation of both the mutant and wild type KRAS alleles, but no change in KRAS copy
number [Figure 6] . In addition, several acquired mutations may contribute to MEKi resistance in these
[11]
cells, including MEK1 G128D mutation that most likely disrupts MEKi binding , and GNAI1 H322N , a Giα1
[31]
subunit of heterotrimeric GTPases that may promote the activity of ERK1/2 and other signalling cascades .
[32]
Selumetinib-resistant LoVo (L6244-R) cells also exhibited parental ERK1/2 activation in the presence of
selumetinib and hyperactivation in the absence of MEKi, both in non-clonal and 12 clonal derivative cell
lines . As with H6244-R, L6244-R cells also proliferated normally in the absence of MEKi, although distinct
[11]
populations did exhibit different degrees of partial reversion to selumetinib sensitivity upon longer-term
drug withdrawal .
[11]

Thus the hyperactivation of ERK1/2 following MEKi withdrawal had no apparent detrimental effect on the
fitness of MEKi-resistant cells with KRAS G13D -amplification/upregulation, which likely underlies the long-
term stability of MEKi resistance in the absence of drug in these models. However, H6244-R and L6244-R
cells did exhibit striking changes in cell morphology when deprived of MEKi; cells exhibited elongated
protrusions, fewer cell-cell contacts, grew over one another and were more motile; all changes consistent
with an epithelial-to-mesenchymal transition (EMT) . Loss of CDH1 (E-cadherin) and increased VIM
[11]
(vimentin) mRNA and protein expression confirmed that these cells had undergone an EMT, and this was
associated with increased expression of SNAI1 (Snail), SNAI2 (Slug) and/or ZEB1 , transcription factors
[11]
known to promote the mesenchymal phenotype and repress CDH1 transcription . These changes following
[33]
MEKi withdrawal could be prevented using the ERK1/2 inhibitor SCH772984, but not PI3K inhibitors,
demonstrating that KRAS amplification acted through ERK1/2, but not PI3K, to drive EMT . Single or double
[11]
siRNA-mediated knock-down of SNAI1, SNAI2 and/or ZEB1 in H6244-R or L6244-R cells demonstrated that
repression of CDH1 by ERK1/2 activation was in large part dependent on ZEB1 [Figure 5]. Indeed, ERK2
[11]
has been shown to promote ZEB1 mRNA and protein expression and EMT in a FRA1-dependent manner .
[34]
In addition, ERK1/2 can promote recruitment of the transcriptional co-repressor CtBP to ZEB1, thereby
silencing CDH1 transcription . Although TWIST1 mRNA and protein expression are positively regulated
[35]
by ERK1/2 in melanoma , there was little change in TWIST1 mRNA or protein expression upon ERK1/2
[36]
hyperactivation in H6244-R or L6244-R cells .
[11]
EMT has been implicated in promoting tumour invasion and metastasis [33,37] . However, in xenograft
experiments there was no evidence of increased invasion into adjacent fat or muscle tissue when H6244-R
tumours were withdrawn from selumetinib, and we could not detect liver or lung metastases in any
condition . These results may be cell line-specific or reflect the limitations of subcutaneous rather than
[11]
orthotopic xenografts; attempts at orthotopic xenotransplantation were hindered by technical difficulties .
[11]
However, whilst the importance of EMT in promoting metastasis has recently been questioned, growing
evidence supports a role in conferring chemoresistance [38,39] . Consistent with this, H6244-R cells that
had undergone EMT in vitro were resistant to 5-fluorouracil (5-FU) and oxaliplatin, standard of care
chemotherapies used to treat colorectal cancer . L6244-R cells that had undergone EMT were also resistant
[11]
to 5-FU, albeit more modestly.