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Identification of Mutant K-Ras-dependent Phenotypes Using
a Panel of Isogenic Cell Lines
*
□
S
Received for publication, June 20, 2012, and in revised form, November 23, 2012 Published, JBC Papers in Press, November 27, 2012, DOI 10.1074/jbc.M112.394130
Steffan Vartanian
‡
, Carolyn Bentley
‡
, Matthew J. Brauer
§
,LiLi
§
, Senji Shirasawa
¶
, Takehiko Sasazuki
储
,
Jung-Sik Kim**, Pete Haverty
§
, Eric Stawiski
‡‡
, Zora Modrusan
‡‡
, Todd Waldman**, and David Stokoe
‡1
From the Departments of
‡
Discovery Oncology,
§
Bioinformatics, and
‡‡
Molecular Biology, Genentech Inc., South San Francisco,
California 94080, the
¶
Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka 814-0180, Japan, the
储
Institute for Advanced Study, Kyushu University, Fukuoka 812-8581, Japan, and the **Department of Oncology and Tumor
Biology Training Program, Lombardi Comprehensive Cancer Center, Georgetown University School of Medicine,
Washington, D. C. 20037
Background: Many studies analyzing K-Ras function rely on overexpression.
Results: Knock-out and knock-in of endogenous mutant K-ras have modest effects on downstream signaling but strong effects
on gene expression and transformation.
Conclusion: Genomic manipulation allows physiological determination of WT and mutant K-Ras consequences.
Significance: Gene expression patterns can be used to monitor inhibition of mutant K-Ras.
To assess the consequences of endogenous mutant K-Ras, we
analyzed the signaling and biological properties of a small panel
of isogenic cell lines. These include the cancer cell lines DLD1,
HCT116, and Hec1A, in which either the WT or mutant K-ras
allele has been disrupted, and SW48 colorectal cancer cells and
human mammary epithelial cells in which a single copy of
mutant K-ras was introduced at its endogenous genomic locus.
We find that single copy mutant K-Ras causes surprisingly mod-
est activation of downstream signaling to ERK and Akt. In con-
trast, a negative feedback signaling loop to EGFR and N-Ras
occurs in some, but not all, of these cell lines. Mutant K-Ras also
had relatively minor effects on cell proliferation and cell migra-
tion but more dramatic effects on cell transformation as
assessed by growth in soft agar. Surprisingly, knock-out of the
wild type K-ras allele consistently increased growth in soft agar,
suggesting tumor-suppressive properties of this gene under
these conditions. Finally, we examined the effects of single copy
mutant K-Ras on global gene expression. Although transcrip-
tional programs triggered by mutant K-Ras were generally quite
distinct in the different cell lines, there was a small number of
genes that were consistently overexpressed, and these could be
used to monitor K-Ras inhibition in a panel of human tumor cell
lines. We conclude that there are conserved components of
mutant K-Ras signaling and phenotypes but that many depend
on cell context and environmental cues.
Ras was one of the first human oncogenes to be discovered
and remains one of the most frequently mutated genes across
multiple human tumors. Ras is a small membrane-bound GTP-
binding protein that recruits effector proteins such as Raf,
phosphoinositide 3-kinase, and RalGDS when bound to GTP,
resulting in activation of these proteins and their downstream
signaling events. Activation of Ras causes a wide variety of bio-
logical consequences depending on the cell type and stimuli,
including increased cell growth, proliferation, survival, differ-
entiation, and morphogenesis. Ras is able to hydrolyze GTP to
GDP, resulting in its self-inactivation, although this process is
slow and is accelerated by a family of proteins termed GTPase-
activating proteins (GAPs). Intrinsic exchange of GDP to GTP
is also quite slow, and this process is accelerated by a family of
proteins termed guanine nucleotide exchange factors (GEFs).
Through the regulation of GAP and GEF activities, the GTP
levels of Ras proteins are carefully adjusted depending on envi-
ronmental conditions, resulting in the maintenance of appro-
priate cellular homeostasis. Mutations of Ras that occur in
human tumors disrupt this process by preventing both intrinsic
and GAP-mediated hydrolysis, causing constitutive GTP asso-
ciation and uncontrolled signaling and proliferation (for
review, see Ref. 1).
Three ras genes are frequently mutated in human tumors,
with characteristic frequencies in different tissues. H-ras is
mutated in bladder cancer (⬃10%) and salivary gland tumors
(⬃15%), N-ras is mutated in melanoma (⬃20%) and hemato-
poietic tumors (⬃15%), and K-ras is predominantly mutated in
pancreatic ductal adenocarcinoma (⬃60%), colorectal cancer
(⬃30%), non-small cell lung adenocarcinoma (⬃20%), and
endometrial cancer (⬃15%) (frequencies as documented on the
Sanger Center COSMIC website). ras mutations can be both
prognostic for overall poor survival (2), as well as predictive for
lack of response to chemotherapy and targeted therapies (3),
showing the clinical importance of this gene. Genetically engi-
neered mouse models have also confirmed that a single point
mutation in K-ras or N-ras expressed in the relevant tissue is
sufficient to develop disease that strongly resembles human
tumors (4 – 6).
In addition to the well described pro-tumorigenic effects of
mutant Ras alleles, there is some suggestive evidence that the
* S. Vartanian, C. Bentley,L. Li, M. J. Brauer, P. Haverty, E. Stawiski, Z. Modrusan,
and D. Stokoe are paid employees of Genentech.
□
S
This article contains supplemental Table 1, Figs. 1– 8, and Materials and
Methods.
1
To whom correspondence should be addressed: Dept. of Discovery Oncol-
ogy, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080. Tel.: 650-
225-6031; Fax: 650-225-6412; E-mail: stokoe.david@gene.com.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 4, pp. 2403–2413, January 25, 2013
© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
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wild type (WT) copy of Ras may possess tumor-suppressive
effects. This was first noticed when mice expressing one copy of
K-Ras generated more and larger lung tumors following chemical
carcinogen treatment than mice expressing both alleles (7). A
tumor-suppressive effect for N-Ras has also been noted in some
mouse tumor models although this seems to be context-depen-
dent (for review, see Ref. 8). Such a tumor-suppressive effect of
WT Ras alleles is not as well characterized in human cancers.
The ability of mutant ras genes to activate downstream sig-
naling pathways that contribute to cell transformation is mostly
undisputed, although almost all of the studies drawing this con-
clusion have been performed using model systems that vastly
overexpress the mutant ras gene, usually involving H-Ras.
However, it has more recently become clear that overexpres-
sion of mutant ras has qualitatively different consequences
compared with single copy gene mutations that presumably
arise during the early stages of human tumorigenesis and that
cell type also plays an important role in determining cellular
responses. For example, overexpression of HRasV12 in immor-
talized mouse NIH3T3 cells causes transformation associated
with activation of Raf and PI3K pathways (9), whereas overex-
pression of HRas in normal fibroblasts causes a senescent-like
cell cycle arrest (10). In contrast, knock-in of a single copy of
mutant K-ras into nontransformed mouse or human cells
causes only very modest consequences on downstream signal-
ing (11–14). The consequences on cell transformation (15) or
tumor formation (11, 12) can also be surprisingly mild in the
absence of additional genetic alterations. Moreover, the assump-
tion of the strong transforming potential of mutant Ras needs to be
reassessed in light of recent discoveries that certain developmental
disorders such as Costello syndrome, cardiofacial cutaneous syn-
drome, and Noonans syndrome are caused by germ line-activating
mutations in ras (for review, see Ref. 16).
In this study, we examined the consequences of single copy
K-ras mutations in the context of human cancer cell lines, as
well as in immortalized but nontransformed human mammary
epithelial cells (HMECs).
2
We utilized cell line derivatives in
which the mutant or WT K-ras alleles had been deleted using
targeted homologous recombination, or in which a single copy
of mutant K-ras had been introduced using the same technol-
ogy. We found that although mutant K-Ras has strong effects
on cellular RasGTP levels, it has surprisingly mild conse-
quences on downstream signaling through Raf and PI3K path-
ways. Mutant K-Ras is also able to initiate negative feedback
signaling to the EGF receptor, which may have relevance in the
response of K-Ras mutant tumors to EGFR inhibitors. Despite
mild consequences on cellular signaling, flux through these
pathways is likely altered as demonstrated by robust regulation
of a small number of genes that seem to be consistently up-reg-
ulated by mutant K-Ras. We also demonstrate that the WT
K-Ras allele may behave in a tumor-suppressive manner in the
cancer cell lines analyzed.
EXPERIMENTAL PROCEDURES
Cell Culture and Lysate Preparation—Hec1A, HCT116, and
SW48 cells were cultured in McCoy’s 5A (Invitrogen) with 10%
FBS and 2 m
ML-glutamine. DLD1 cells were grown in RPMI
1640 medium with 10% FBS and 2 m
ML-glutamine, and
HMECs were cultured in 50:50 DMEM/F12 with 10% FBS, 20
ng/ml EGF, 0.01 mg/ml insulin, 500 ng/ml hydrocortisone, and
2m
ML-glutamine. Cells were cultured in a 37C/5% CO
2
incu
-
bator. For standard Western blotting and immunoprecipita-
tions, cells were harvested in radioimmuneprecipitation assay
buffer (50 m
M Tris, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1%
Nonidet P-40, 0.1% SDS) with Complete Protease Inhibitor
(Roche Applied Science) and Phosphatase Inhibitor Mixture II
(Sigma). For Ras binding domain (RBD) pulldown experiments,
cells were harvested in 1⫻ MgCl buffer (Millipore) with Com-
plete Protease Inhibitor.
siRNA Transfections—Cells were grown to 80% confluence in
10-cm dishes and transfected with nontargeting control or
K-Ras OTP siRNAs (Dharmacon). Briefly, 900 pmol of siRNA
and 20
l of RNAiMax (Invitrogen) were diluted separately in
1.5 ml of OptiMEM each. The siRNA and lipid were then mixed
together and incubated for 20 min at room temperature. The
complexes (3 ml) were added to the cells and incubated for 72 h.
Immunoprecipitations/Immunoblots—To check for siRNA
silencing of Ras, 2.5 mg of lysate was incubated with 10
lofPan
Ras antibody EP1125Y (Millipore 04-1039) at 4 ºC overnight. 50
l of protein A bead slurry was then added and incubated for 2 h
at 4 ºC with rotation. Beads were washed three times with lysis
buffer and resuspended in sample buffer. RBD pulldowns were
performed using a Ras Activation Assay kit (Millipore 17-218)
according to the manufacturer’s protocol. Western blot sam-
ples were run on 4 –20% Tris-glycine gels and transferred to
PVDF membranes using the iBlot system (Invitrogen). Pan Ras
immunoprecipitations were blotted for K-Ras (Santa Cruz
sc-30). RBD immunoprecipitations were blotted for Pan Ras
(Millipore 05-516), H-Ras, N-Ras, and K-Ras (Santa Cruz sc-29,
sc-31, and sc-30, respectively). Cell lysates were also blotted for
pERK (Cell Signaling 9101s), pAkt (Cell Signaling 9271s), pCbl
(Cell Signaling 3555s), pEGFR-Y1125 (Abgent AP3376a),
pEGFR-Y1069 (Upstate 07-715), total EGFR (Cell Signaling
4405s), total Cbl (Cell Signaling 2179s),

-actin (Sigma A2228),
total Ras (Millipore 05-516), total ERK (Cell Signaling 9102s),
and total Akt (Cell Signaling 4685s). In specified experiments,
cells were treated with GDC0941 (Fisher RG007-25MG) or
PD0325901 (Fisher RP02-10MG) diluted in dimethyl sulfoxide.
Soft Agar Assays—Base agar layer was prepared by mixing
equal parts 2⫻ medium ⫹ 20% FBS with 2.4% agarose. 1 ml of
this base agar was then added to each well of a 12-well tissue
culture plate (Grenier) and placed in a 4 ºC refrigerator to cool.
Cells were trypsinized and counted, then diluted to 5000 cells/
333
lof1⫻ medium ⫹ 10% FBS. A 1:1 mixture of 2⫻ medium ⫹
20% FBS and 2.4% agarose was then prepared, and 667
l was
added to the cells, for a final agarose concentration of 0.8% in
the cell layer. 1 ml of cell mixture was then pipetted on top of
the base agar layer and placed at 4 ºC for 15 min to cool. The
plates were placed in a 37 ºC/5% CO
2
incubator overnight, and
2
The abbreviations used are: HMEC, human mammary epithelial cell; EGFR,
EGF receptor; RBD, Ras binding domain.
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1 ml of medium was added on top of the cell layer the next day.
These assays were set up in triplicate.
Exome Alignment and Variant Calling—Illumina fastq reads
were mapped to the UCSC human reference genome (GRCh37/
hg19) using BWA (17) set to default parameters. Duplicate mark-
ing, local realignment, and variant calling was done as described
previously (18). Variants included in dbSNP Build 131 (19) that
were also absent in COSMIC v56 (20) were excluded.
Proliferation/Migration Assays—For proliferation assays,
cells were trypsinized, counted, and plated at 2500 or 5000 cells
in 150
l/well, in a 96-well clear-bottomed black tissue culture
plate (BD Falcon). Cells were left to proliferate over 7 days in an
Incucyte instrument, which calculates cell density over speci-
fied intervals.
For migration assays, cells were plated the night before at
50,000 cells/well in a fibronectin- or collagen-coated Image-
Lock 96-well plate (Essen Bioscience). Cells were grown to con-
fluence, then a wound was created using the WoundMaker
apparatus (Essen Bioscience) according to the manufacturer’s
recommended protocol. The cells were then washed twice with
PBS and medium replaced to 100
l. Plates were imaged over
24 h at 2-h intervals.
Taqman Assays—RNA was isolated from cells using the
RNeasy kit (Qiagen 74106), and cDNA was synthesized using
the High Capacity cDNA Reverse Transcription Kit (Applied
Biosystems 4368814). Taqman assays were set up in multiplex,
using GAPDH as the endogenous control. The target probes
(DUSP5, DUSP6, ETV1, ETV5, NT5E, UPP1,K-ras, and IER3)
were tagged with FAM dye. Assays were run in 384-well format,
using 20 ng of input cDNA/reaction.
Additional procedures are described in the supplemental
Materials and Methods.
RESULTS
To assess the contribution of single copy K-Ras mutations
across multiple cell lines, we analyzed three previously pub-
lished tumor cell lines in which the wild type or mutant K-ras
allele was disrupted by homologous recombination. These cell
lines are the colorectal cancer lines DLD1 and HCT116 (21, 22),
which express heterozygous G13D mutations, and the endome-
trial cancer line Hec1A (23), which expresses a heterozygous
G12D mutation. In addition, we also analyzed two cell lines in
which a single copy of G13D mutant K-ras was introduced at its
endogenous locus, a colorectal cancer cell line SW48 which
evolved in the absence of mutant Ras, and nontransformed but
immortalized HMECs (15). Whole genome copy number anal-
ysis shows that for the lines analyzed (DLD1 and Hec1A),
although not truly “isogenic,” there are only very few and rela-
tively minor differences at the chromosomal level between the
parental lines and their isogenic derivatives (supplemental Fig.
1).
First we assessed the levels of RasGTP in these cell lines
growing in 10% FBS, under starved conditions, and following
short term stimulation with EGF. The RBD of cRaf was used as
an affinity probe for RasGTP, as described previously (24). In all
cases, knock-out and knock-in of mutant K-Ras decreased and
increased KRasGTP levels, respectively (Fig. 1A). Interestingly,
the presence or absence of mutant K-Ras affected NRasGTP
levels; in HCT116 and SW48 cells, the presence of mutant
K-Ras was associated with increased NRasGTP levels. This
could be due to the previously described positive feedback loop
caused by allosteric activation of the Ras exchange factor sos
(25). In contrast, in Hec1A, HMECs, and to a lesser extent
DLD1 cells, the presence of mutant K-Ras was associated with
decreased NRasGTP levels, suggesting the presence of a nega-
tive feedback loop. We also analyzed the levels of HRasGTP
using the same approach, but we did not detect any H-Ras pres-
ent in the RBD pulldowns under the conditions tested (data not
shown).
The effects of mutant K-Ras on downstream signaling to ERK
and Akt were examined next. In contrast to the dramatic effects
FIGURE 1. Consequences of mutant K-ras knock-out and knock-in on RasGTP levels and downstream signaling. A, cell lines of the indicated genotypes
were grown in 10% FBS, serum-starved for 18 h, or stimulated for 10 min with 100 ng/ml EGF. RasGTP was affinity-purified from 2.5 mg of cell lysate using
GST-Raf-RBD and analyzed by Western blotting with Ras isoform-specific antibodies. In addition, 40
g of lysate was used for Western blotting for total Ras
protein. B, the indicated cell lines were treated as above, and 40
g of protein lysates separated by SDS-PAGE and analyzed by Western blotting with
anti-phospho-ERK, phospho-Akt, total ERK, total Akt, and

-actin antibodies.
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on KRasGTP levels, the presence or absence of mutant K-Ras
had very modest effects on ERK and Akt phosphorylation,
under conditions of either basal proliferation in 10% FBS,
serum starvation, or acute stimulation with EGF (Fig. 1B).
Hec1A cells lacking mutant K-Ras actually showed slightly
increased phosphorylation of these proteins following EGF
stimulation, a paradoxical observation previously noted by
Waldman and colleagues (23).
To better understand the impact of single copy mutant K-Ras
on cell signaling, we performed a global phosphotyrosine anal-
ysis in the Hec1A
G12D/⫺
and Hec1A
⫺/WT
derivative cell lines
using Epitome phosphotyrosine arrays. Similar to the effects
seen by Western blot analysis, EGF-stimulated ERK1 and ERK2
phosphorylation was higher in the KRasG12D-deleted cells
(supplemental Fig. 2 A). The most dramatically altered proteins
between these cell lines were EGFR and its downstream sub-
strate Cbl (Fig. 2A). Both of these proteins showed increased
phosphorylation in response to EGF in the mutant K-Ras
knock-out clones compared with the WT K-Ras knock-out
clone. We confirmed these results by Western blot analysis
(Fig. 2B), which also analyzed the individual phosphorylation
sites, Tyr-1125 (a Grb2 binding site) and Tyr-1069 (a Cbl bind-
ing site). To determine whether the inhibitory effect of mutant
K-Ras on EGFR phosphorylation was a general phenomenon,
we examined the additional cell lines for basal and stimulated
EGFR and Cbl phosphorylation. DLD1 cells lacking mutant
K-Ras also showed increased EGFR phosphorylation at Tyr-
1125, even as total EGFR levels were decreased in this cell line
derivative (Fig. 2B). In addition, Cbl also showed increased
phosphorylation in the K-Ras knock-out DLD1 lines, although
this was more apparent under basal or starved conditions (Fig.
2B). HMECs expressing mutant K-Ras also showed decreased
EGFR phosphorylation (Fig. 2C). Increased EGFR phosphory-
lation was also seen in independently derived Hec1A WT and
mutant K-Ras knock-out clones (supplemental Fig. 2B), show-
ing that these effects are not due to clonal artifacts. However,
neither HCT116 nor SW48 cell isogenic lines showed any obvi-
ous differences in EGFR or Cbl phosphorylation in the K-Ras
knock-out or knock-in derivative cell lines (supplemental Fig. 2,
C and D). Therefore, mutant K-Ras negatively regulates EGFR
phosphorylation, as well as NRasGTP levels, in some cell lines.
To test the hypothesis that signaling downstream of mutant
K-Ras might be responsible for suppressing EGF-induced EGFR
phosphorylation, we utilized small molecule inhibitors of MEK
and PI3K in the mutant K-Ras-expressing cells. Fig. 3A shows that
PD0325901 was able to dramatically increase the response to EGF
in the mut/WT and mut/⫺ Hec1A and DLD1 cells. A smaller
increase was also seen with GDC-0941. PD0325901 also increased
EGFR phosphorylation in the Hec1A
⫺/WT
cell line, although this
was not seen with GDC-0941 or by either compound in the
DLD
⫺/WT
cells. Interestingly, NRasGTP levels were also increased
with these inhibitors, suggesting a mechanistic link between EGFR
phosphorylation and NRasGTP levels. To determine the nature of
mutant K-Ras-induced EGFR inhibition, we asked whether condi-
tioned media from mut/⫺ cells could inhibit EGFR phosphoryla-
tion in ⫺/WT cells. Supplemental Fig. 3 shows that there is a slight
inhibition of EGFR phosphorylation in cells in Hec1A
⫺/WT
cells
using conditioned media from Hec1A
mut/⫺
cells, but not in the
FIGURE 2. Mutant KRas negatively regulates EGFR signaling. A, Hec1A cells deleted for either WT or mutant KRas were serum-starved or starved and
stimulated with 100 ng/ml EGF for 10 min. 1 mg of lysate was reduced, alkylated, digested with Lys-C, and used to probe antibody microarrays. Signal for EGFR
and Cbl phosphorylation (mean ⫾ S.D. (error bars, n ⫽ 3)) is shown. B, the indicated cell lines were cultured in 10% FBS, 0% FBS, or stimulated with 100 ng/ml
EGF, and 40
g of protein lysate was separated by SDS-PAGE and probed by Western blotting using the antibodies shown. C, results are the same as for B but
using HMECs of the indicated genotypes.
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similar DLD1 cell conditioned media swap. These results suggest
that activation of ERK downstream of mutant K-Ras results in a
signal that antagonizes response to EGF, which is partially (for
Hec1A cells) as a result of a secreted factor. The use of a cytokine
array did not help to clarify which secreted factor might be respon-
sible for this (data not shown).
We next examined the effects of endogenous mutant K-Ras
on cellular phenotypes. Cell proliferation was not dramatically
affected in any of the cell lines examined, although DLD1
⫺/WT
cells showed slightly decreased proliferation relative to parental
and DLD1
G12D/⫺
clones, and SW48 cells showed slightly
increased proliferation when mutant K-Ras was introduced
(Fig. 4A). We then analyzed cell migration using a classic
scratch wound-healing assay. DLD1 isogenic cell lines cells
showed little consistent differences in cell migration, whereas
Hec1A cells showed a slight decrease in migration upon loss of
the mutant K-ras allele, but increased migration upon loss of
the WT K-ras allele. Knock-in of mutant K-ras strongly
decreased the migration of HMECs, which showed the fastest
migration capacity of the cell types examined, with the wound
being completely closed in 20 h (Fig. 4B). The migration of
SW48 and HCT116 cells could not be assessed using this assay,
as HCT116 cells did not remain attached to the plate during the
wound-making process, and SW48 cells did not form a suffi-
ciently confluent monolayer.
We analyzed the transforming capacity of these cell lines
using anchorage-independent growth in soft agar. As expected,
and as previously reported, deletion of the mutant K-ras allele
strongly decreased the number of colonies in soft agar in DLD1,
HCT116, and Hec1A cells (Fig. 4C). There was also a modest
increase in the number of colonies in the HMECs expressing
mutant K-Ras, although the number of colonies in this line was
lower than seen in the cancer cell lines. Interestingly, the num-
ber of colonies was significantly increased in all three cell lines
lacking the wild type K-ras allele. SW48 cells did not form col-
onies under these conditions in our hands.
To attempt to understand why deletion of the WT K-ras
allele could enhance soft agar growth, we compared the signal-
ing pathways affected by this in Hec1A and HCT116 cells, the
cells that were most dramatically affected in this assay. Supple-
mental Fig. 4 shows that of 45 phosphorylation sites on signal-
ing proteins, only P-ERK (in Hec1A cells) and P-Akt (in
HCT116 cells) showed any differences. To determine the fre-
quency of WT K-ras allele loss in human tumor cell lines, we
assessed the K-ras mutant allele frequency in a panel of 624 cell
lines which had been subjected to exome-capture and deep
sequencing.
3
Of 130 cell lines with KRas variants, 35 showed
KRas variant DNA read frequencies consistent with homozy-
gous mutant K-ras status (Table 1). Moreover, even human
tumor cell lines classified as heterozygous for mutant K-ras
DNA frequently showed preferential expression of the mutant
K-ras allele, as assessed by combined exome-seq and RNAseq
analysis (Fig. 5). Combined, these data suggest that there is
selective pressure to lose or reduce expression of the WT K- ras
allele, at least in human cancer cell lines.
To address whether the signaling and phenotypic conse-
quences of chronic loss of mutant K-ras are contributed by
either clonal anomalies or long term adaptation, we also ana-
lyzed the effects of short term K-Ras knockdown using siRNA.
Knockdown of K-Ras (WT and mutant) in these cell lines
showed similar modest effects on ERK and/or Akt phosphory-
lation in the basal and starved settings (Fig. 6, A–C). Interest-
ingly, the increased EGFR phosphorylation seen in the K-Ras
knock-out DLD1 and Hec1A cells was also seen following
K-Ras knockdown, although the magnitude of this effect was
diminished (Fig. 6, A and C). Transient knockdown of K-Ras in
HCT116 cells also increased EGFR phosphorylation, despite
incomplete knockdown efficiency in this cell line (Fig. 6B). The
effects of K-Ras knockdown on proliferation and migration
were also assessed. K-Ras knockdown had little effect on pro-
liferation in any cell line (supplemental Fig. 5A), similar to the
results with the mutant K-Ras knock-out derivatives. Knock-
down of K-Ras in DLD1 cells had little effect on migration,
whereas K-Ras knockdown decreased migration of Hec1A cells
(supplemental Fig. 5B).
We then asked whether a combined analysis of K-Ras
isogenic cell lines could be used to identify genes that are
commonly regulated by mutant K-Ras expression. We pro-
filed the Hec1A, DLD1, and SW48 isogenic cell lines on
Affymetrix human genome U133 Plus microarrays. Knock-
out or knock-in of K-ras did not have strong effects on global
gene expression, cell lines clustered by parental cell line and
not K-Ras genotype (Fig. 7A). Analysis of multiple individual
clones of the same genotype (only performed for Hec1A
cells) showed that there were only small differences that
could be attributed to clone-specific alterations and that
independent genotypically matched lines clustered together.
3
C. Klijn, E. Stawiski, Z. Zhang, S. Seshagiri, and members of the Genentech
Cell Line Genomics Group, unpublished findings.
FIGURE 3. Signaling through the MEK and PI3K pathways negatively regulates EGFR and NRas activity. Hec1A and DLD1 cells of the indicated genotypes
were plated in 6-well dishes and at 70% confluence treated with the indicated concentration of the MEK inhibitor PD0325901 and the PI3K inhibitor GDC-0941.
18 h later, the cells were lysed, and 40
g was subjected to SDS-PAGE and Western blotting using the antibodies shown.
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Interestingly, WT K-ras-deleted lines clustered more closely
to parental cell lines than mutant Kras-deleted lines, sug-
gesting that in each cell line the mutant KRas allele has a
stronger effect on global gene expression than the WT allele.
The lists of genes affected by mutant K-Ras expression in
these three cell lines are shown in supplemental Table 1.In
general, the spectrum of genes regulated by the presence or
absence of mutant K-Ras in the three different cell lines was
quite distinct, as seen by overlaying the most significant up-
and down-regulated genes from DLD1 cells onto Volcano
FIGURE 4. K-Ras status has modest effects on cell proliferation and migration but more dramatic effects on colony formation. A, the indicated isogenic
cell lines were plated in 96-well plates at 5000 cells/well in 10% FBS, and confluence was tracked over 7 days using an Incucyte instrument. The means ⫾ S.D.
(error bars, n ⫽ 3) at 2-h intervals are shown. B, cell migration was analyzed by creating a scratch in the confluent cell monolayer and following wound closure
over 24 h using an Incucyte instrument. The left panel shows the percentage wound closure measured at 2-h intervals (mean ⫾ S.D., n ⫽ 3), and the right panels
show images of the wound at 0 and 24 h. C, the isogenic cell lines were seeded in a 0.8% agar/growth medium layer, and colony formation was analyzed after
30 days. The graphs show the average ⫾ S.D. of triplicate wells counted using a GelCount instrument, and the images show representative sections of one well.
TABLE 1
Frequency of heterozygous (Het) and homozygous (Hom) K-ras mutations in a panel of cell lines
Gene Mutation Count Het/Hom (count) Tissue (counts)
Average variant
allele frequency
K-ras G12D 40 Het (37), Hom (3) Stomach (1), lung (2), pancreas (19),
uterus (10), ovary (2), colon (5),
breast (1)
0.52
K-ras G12V 22 Hom (9), Het (13) Lung (4), colon (4), pancreas (13),
breast (1)
0.75
K-ras G12C 16 Hom (9), Het (7) Lung (11), rectum (1), colon (2),
ovary (1), urinary bladder (1)
0.80
K-ras G13D 15 Het (14), Hom (1) Lung (4), breast (1), colon (6),
blood (1), ileum (1), lymph node (2)
0.43
K-ras G12R 7 Het (6), Hom (1) Pancreas (5), skin (1), breast (1) 0.65
K-ras G12A 6 Hom (1), Het (5) Blood (4), lung (1), colon (1) 0.54
K-ras Q61H 5 Hom (5) Lung (3), kidney (1), pancreas (1) 1
K-ras G13C 4 Hom (1), Het (3) lung (3), ovary (1) 0.61
K-ras A146T 3 Het (2), Hom (1) Blood (1), bone marrow (1), cecum (1) 0.67
K-ras G12S 2 Hom (1), Het (1) Lung (1), bone (1) 0.69
K-ras Q61K 2 Hom (1), Het (1) Stomach (1), lung (1) 0.83
K-ras Q61L 2 Hom (1), Het (1) Lung (1), colon (1) 0.79
K-ras A146V 1 Het (1) Blood (1) 0.75
K-ras A59T 1 Het (1) Bone (1) 0.45
K-ras K117N 1 Hom (1) Blood (1) 1
K-ras L19F 1 Het (1) Lung (1) 0.51
K-ras P121H 1 Het (1) Ovary (1) 0.52
K-ras Y64C 1 Het (1) Colon (1) 0.48
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plots of gene expression in the three cell lines (Fig. 7B). Sim-
ilar disparate observations were also seen with the up- and
down-regulated genes from Hec1A and SW48 cells (supple-
mental Fig. 6). Nevertheless, we searched for genes that dis-
played common regulation in response to mutant K-Ras and
found two genes (NT5E and ETV1) that were commonly up-
regulated by mutant K-Ras in all three cell lines (Fig. 7C). We
also found several genes that were commonly up-regulated
by mutant K-Ras in Hec1A and DLD1 cells, including ETV5,
UPP1, IER3, DUSP5, and DUSP6.
We confirmed the mutant K-Ras-dependent regulation of
these genes by Taqman analysis in these cell lines (Fig. 8A). All
genes showed similar regulation by Taqman compared with the
array; in addition although IER3, ETV5, and UPP1 were not
identified as up-regulated by mutant K-Ras in SW48 cells by
microarray, they did show up-regulation by Taqman. We next
asked whether these genes could more generally be used as
markers for K-Ras inhibition in a panel of K-Ras mutant cell
lines and additionally asked whether they were also affected by
K-Ras knockdown in WT K-Ras cell lines. K-Ras siRNA effec-
tively decreased K-Ras expression in all of the lines examined
(although knockdown was modest in HupT3 and HT55 cells)
and decreased P-ERK in the mutant K-Ras-expressing cell lines
(Fig. 8B). P-ERK was not affected in the K-Ras WT cell lines, nor
was P-Akt decreased in any cell line. The basal expression of
these genes in this panel of cell lines did not correlate with
mutant K-Ras status (data not shown). However, all of the
mutant K-Ras-specific genes identified from the isogenic cell
lines were generally decreased following K-Ras knockdown in
the mutant K-Ras-expressing cell lines. In contrast, their
expression in the WT K-Ras cells was much less affected, con-
firming their usefulness as general markers for inhibition of
mutant K-Ras signaling (supplemental Fig. 7 and summarized
in Fig. 8C). This effect was statistically significant for all genes
apart from ETV1, which had low/undetectable expression in
this experiment in two cell lines.
To determine whether any of the genes induced by mutant
K-Ras contributes to its transformation properties, we knocked
down ETV1 and NT5E using siRNA pools in the same panel of
11 cell lines, and monitored proliferation in the second dimen-
sion on plastic and in the third dimension in hanging drop cul-
tures (26). Knockdown relative to a nontargeting control is
shown in supplemental Fig. 8, A–C. Whereas K-Ras knock-
down inhibited proliferation in both the second and third
dimensions selectively in the mutant K-Ras-expressing cell
lines, there was little selective inhibition of proliferation follow-
ing knockdown of either NT5E or ETV1 (supplemental Fig. 8, D
and E).
DISCUSSION
K-Ras is widely recognized as an important human oncogene
that is mutated in a large percentage of human epithelial
tumors. However, most of the analyses of the cellular conse-
quences of Ras expression have been performed using overex-
pression studies, often using H-Ras, and frequently using cell
types that are either mesenchymal or murine. In this study, we
have documented the effects of the endogenous mutated K-ras
allele, using cell line derivatives generated by homologous
recombination targeting. These studies have demonstrated
that there are both common phenotypes to mutant K-Ras
expression across multiple cell lines, as well as consequences
that are unique to the cell line, and therefore likely also unique
to the tumor tissue from which these were originally derived.
The use of isogenic cell lines has several advantages over com-
monly utilized overexpression and knockdown approaches.
Overexpression of mutant ras alleles results in a senescent-like
phenotype that has been attributed to increased production of
reactive oxygen species and associated stresses (27, 28) and is
likely unrelated to the normal functions of single copy mutant
ras, which results in tumor initiation without senescence
(11). The use of RNAi against ras (similar to any gene knock-
down) is problematic due to incomplete knockdown and the
possibility of off-target effects associated with siRNA or shRNA
sequences. Moreover, subtleties associated with activities of the
remaining WT allele would also not be revealed using a conven-
tional knockdown approach. The concerns of artifactual results
due to cell line selection and clonal anomalies were partially
alleviated in this study by the use of multiple clones (where
available) and the use of transient knockdown to show similar
effects on signaling and biological phenotypes.
One common consequence of mutant K-ras deletion was the
detrimental effects on soft agar colony growth, an often used
surrogate assay for tumorigenesis. This shows that, even with
the multitude of genetic alterations present in these cell lines,
deletion of a single allele has substantial effects on transforma-
tion. In contrast to the strong effect in transformation assays,
deletion of mutant K-ras has mild or nonexistent effects on cell
proliferation under the conditions assessed. This is unlikely to
be a trait selected for during the generation of these lines, as
knockdown of K-Ras in the parental lines also had only modest
effects on cell proliferation. Recently, there has been an appre-
ciation that K-Ras mutant cell lines vary in their response to
K-Ras knockdown, with some cell lines showing a strong
decrease in cell number due to enhanced apoptosis and/or
FIGURE 5. WT K-ras is either deleted or underexpressed in the majority of
mutant KRas-expressing cell lines. The read counts of WT (y axis) and
mutant (x axis) K-ras using RNA-seq data from 88 mutant K-Ras cell lines are
plotted. Lines designated as homozygous mutant K-Ras are shown in blue;
lines designated as heterozygous mutant K-Ras are shown in red.
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growth arrest, and other cell lines showing little effect (29, 30).
In these published studies, Hec1 (of which Hec1A is a deriva-
tive), HCT116, and DLD1 cells were all predicted and empiri-
cally shown to be insensitive to K-Ras knockdown in prolifera-
tion assays, consistent with the data shown here. It perhaps is
not surprising that the isogenic K-Ras lines that exist are “insen-
sitive,” as K-Ras-“sensitive” lines would likely be unable to
expand following deletion of the mutant allele. Nevertheless,
we would suggest that even though a cell line is classified as
Ras-independent by proliferation criteria, it remains Ras-de-
pendent by more stringent growth conditions that may more
closely reflect the tumor environment.
FIGURE 6. K-Ras siRNA knockdown in parental cell lines shows similar effects as chronic mutant K-Ras deletion. A, 100 nM K-Ras siRNA pools were
transfected into DLD1 cells and treated as shown. 72 h following transfection, 2.5 mg of protein lysates was subjected to pulldown with GST-RBD glutathione
bead slurry, and total RasGTP and NRasGTP were analyzed by Western blotting with the indicated antibodies. Additionally, 40
g of protein lysate was
separated by SDS-PAGE and analyzed by Western blotting using the indicated antibodies. B and C, HCT116 and Hec1A cells were treated as described in A.
FIGURE 7. Mutant K-Ras initiates distinct gene transcription programs that are generally cell line-dependent. A, RNA from triplicate cultures of the
indicated cell lines was hybridized onto Affymetrix U133 microarrays. The 403 genes that were differentially expressed in at least two cell lines were clustered
using the complete linkage method as implemented in the function hclust. B, genes that were regulated by mutant K-Ras in DLD1 cells were overlaid onto all
genes differentially regulated by mutant K-Ras in DLD1 cells (left plot), Hec1A cells (middle plot), and SW48 cells (right plot). C, genes up-regulated or down-
regulated by mutant K-Ras expression ⬎2-fold in different cell lines are shown.
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One aspect of mutant K-Ras signaling that was common to
some, but not all, of the cell lines studied here, was the cross-
talk between K-Ras and N-Ras. This was most dramatically seen
in the Hec1A cells and HMECs, but was also seen in DLD1 cells
to a lesser extent. This also correlated with the ability of mutant
K-Ras to down-regulate EGFR phosphorylation, an observation
also recently made by in lung cancer cell lines (31) and in colo-
rectal cancer cell lines (32). This could explain the relatively
mild effects on downstream signaling in the different cell lines
upon mutant K-ras deletion or expression, i.e. increased NRas-
GTP might compensate for the loss of KRasGTP and in the case
of Hec1A cells, could explain the paradoxical increase in EGF-
stimulated ERK phosphorylation upon mutant K-ras deletion.
This observation may also contribute to the lack of effect of
EGFR inhibitors in mutant K-Ras lung and colorectal tumor
patients (33, 34). Although activated Ras could maintain down-
stream signaling in the presence of EGFR inhibition, it could
also shut off EGFR signaling in these tumors, rendering them
independent of EGF and therefore refractory to EGFR inhibi-
tion. Here we show that the decreased EGFR phosphorylation
and NRasGTP levels in mutant K-Ras-expressing cell lines are
largely due to negative feedback initiated by MEK signaling.
This could be mediated, at least in part, by production of
secreted factors that antagonize EGF-dependent signaling,
although we were unable to define the exact factor that causes
this.
Deletion or introduction of mutant K-ras caused surprisingly
diverse effects on gene expression, showing that mutant K-Ras
has specific functions depending on the tissue in which it is
expressed. There have previously been several publications
using “Ras gene signatures” to classify human tumors. Sweet-
Cordero et al. (35) analyzed mutant K-Ras-driven lung tumors
and compared them with normal mouse lung tissue and human
K-Ras mutant lung cancers to derive 89 genes up-regulated in
mutant K-Ras lung cancer. Bild et al. (36) overexpressed H-Ras
in HMECs to derive 177 genes up-regulated by H-Ras. Arena et
al. (37) used single copy K-Ras knock-in in mouse liver progen-
itor cells to derive 149 genes that were up-regulated upon this
condition. Mutant K-Ras results in highly divergent conse-
quences in different tissues and environments, as exemplified
by the fact that only a single gene (DUSP6) is in common among
these three sets of signatures. Although the approach used here
to identify mutant K-Ras-regulated genes is likely no better or
no worse, it confirms that only a small number of genes are
consistently altered by mutant K-Ras. Notably, the seven genes
identified and validated in this study have all been found asso-
ciated with Ras expression or signaling in previous studies;
DUSP6, DUSP5 (36, 38), NT5E, ETV1 (39), IER3 (36, 39), UPP1
(36, 40), and ETV5 (39, 41). The functions of these genes are
quite diverse and represent mechanisms involved in negative
feedback signaling (DUSP5 and 6), nucleoside metabolism
(NT5E and UPP), transcriptional responses activated down-
stream of ERK signaling (ETV1 and ETV5), and protection from
apoptosis (IER3). Knockdown of NT5E has previously been
shown to decrease transformation properties of K-Ras mutant
MDA-MB-231 cells (42). Although knockdown of NT5E did
decrease proliferation in the second and third dimensions in
K-Ras mutant A427 cells, there was no statistically significant
correlation between the effects in mutant versus WT K-Ras
cells (supplemental Fig. 6). It is likely that K-Ras uses multiple
target genes to fully elicit transformation properties.
FIGURE 8. Expression of 7 genes are commonly regulated by mutant K-Ras. A, RNA was purified from the indicated cell lines and subjected to Taqman
quantitative PCR analysis against the genes shown in each graph. Expression was normalized to the control gene GAPDH and expressed (mean ⫾ S.D. (error
bars), n ⫽ 3) relative to the levels seen in the parental cell line. B, a panel of 11 cell lines expressing WT or mutant K-Ras was treated with 100 n
M NTC or K-Ras
siRNA pools, and protein and RNA were harvested 72 h later from identically treated plates. K-Ras knockdown was assessed by immunoprecipitation-Western
blotting using total and K-Ras-specific antibodies, and expression of P-ERK and P-Akt was assessed by Western blotting. C, RNA isolated from NTC or K-Ras
siRNA-treated cells was subjected to Taqman quantitative PCR using the indicated probes, and the effects on gene expression in each cell line are indicated as
a ratio of K-Ras:NTC siRNA treatment. The horizontal bar indicates the average expression of the gene across all cell lines. The open symbols represent WT
K-Ras-expressing cells, and the filled symbols represent mutant K-Ras-expressing cells. p values are the results of an unpaired t test comparing the effects on
mutant versus WT K-Ras-expressing cells.
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The increased transformation properties of Hec1A, HCT116, and
DLD1 cells lacking the WT K-ras allele was unexpected but is
reminiscent of studies in mice suggesting that the WT K-ras
allele can display tumor-suppressive effects. This was first
demonstrated in chemical carcinogen-induced tumors that
result in K-ras mutation, that were more aggressive when one
copy of K-ras was deleted in the germ line (7). Loss of the wild
type K-ras allele was also recently observed in high grade, but
not low grade lung tumors driven by conditional mutant K-Ras
(43). A tumor-suppressive function for WT N-Ras has also been
observed in mouse tumors driven by both chemical carcino-
gens, as well as by transgenic expression of mutant N-Ras (44).
The potential for WT K-Ras to act as a tumor suppressor in
human tumors has not been as well characterized, although our
analysis of a large panel of human cancer cell lines shows that
⬎one-quarter of mutant K-Ras-expressing cells have allelic
imbalance/homozygous expression of the mutant allele. Even
in cells that have heterozygous expression, a majority show
enhanced expression of the mutant allele. These results clearly
suggest a selective advantage for the tumor cells to lose the WT
K-ras allele or to decrease its expression. The model systems
used in this study, or other similar ones, should allow further
dissection of this unexpected phenomenon.
Acknowledgments—We thank the members of the sequencing, bioin-
formatics, cell culture, and microarray core facilities at Genentech for
expertise and timely analysis; Chris Klijn and Zemin Zhang for K-Ras
RNA-seq information; Fred de Sauvage and Jeff Settleman for critical
input during this project; and David Davis for many useful and
thoughtful discussions.
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Mutant Ras-dependent Signaling and Gene Expression
JANUARY 25, 2013 • VOLUME 288 •NUMBER 4 JOURNAL OF BIOLOGICAL CHEMISTRY 2413
by guest on October 27, 2015http://www.jbc.org/Downloaded from
David Stokoe
andStawiski, Zora Modrusan, Todd Waldman
Sasazuki, Jung-Sik Kim, Pete Haverty, Eric
J. Brauer, Li Li, Senji Shirasawa, Takehiko
Steffan Vartanian, Carolyn Bentley, Matthew
Lines
Phenotypes Using a Panel of Isogenic Cell
Identification of Mutant K-Ras-dependent
Signal Transduction:
doi: 10.1074/jbc.M112.394130 originally published online November 27, 2012
2013, 288:2403-2413.J. Biol. Chem.
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