Activation of KRAS Promotes The Mesenchymal
Activation of KRAS Promotes The Mesenchymal
Activation of KRAS Promotes The Mesenchymal
99
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ORIGINAL ARTICLE
Basal-type breast cancers are among the most aggressive and deadly breast cancer subtypes, displaying a high metastatic ability
associated with mesenchymal features. However, the molecular mechanisms underlying the maintenance of mesenchymal
phenotypes of basal-type breast cancer cells remain obscure. Here, we report that KRAS is a critical regulator for the
maintenance of mesenchymal features in basal-type breast cancer cells. KRAS is preferentially activated in basal-type breast
cancer cells as compared with luminal type. By loss and gain of KRAS, we found that KRAS is necessary and sufficient for the
maintenance of mesenchymal phenotypes and metastatic ability through SLUG expression. Taken together, this study
demonstrates that KRAS is a critical regulator for the metastatic behavior associated with mesenchymal features of breast cancer
cells, implicating a novel therapeutic target for basal-type breast cancer.
Experimental & Molecular Medicine (2015) 47, e137; doi:10.1038/emm.2014.99; published online 30 January 2015
1
Department of Life Science, Research Institute for Natural Sciences, Hanyang University, Seoul, Korea; 2Laboratory of Radiation Exposure & Therapeutics,
National Radiation Emergency Medical Center, Korea Institute of Radiological and Medical Sciences, Seoul, Korea and 3Department of Radiation Biology,
Environmental Radiation Research Group, Korea Atomic Energy Research Institute, Daejeon, Korea
4These authors contributed equally to this work.
Correspondence: Professor S-J Lee, Laboratory of Molecular Biochemistry, Department of Life Science, Hanyang University, 17 Haengdang-Dong,
Seongdong-Ku, Seoul 133-791, Korea.
E-mail: sj0420@hanyang.ac.kr
Received 27 August 2014; revised 15 October 2014; accepted 28 October 2014
KRAS promotes mesenchymal features
R-K Kim et al
basal- and luminal-type of breast cancer cells, we show that the 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 1 mM Na3VO4, 10%
activity of KRAS is preferentially higher in basal-type breast glycerol, 10 μg ml − 1 leupeptin, 10 μg ml − 1 aprotinin and 25 mM NaF),
cancer cells compared with the luminal type. We also the activated KRAS (GTP-RAS) bound to RAF-1 RBD argarose beads
demonstrate that KRAS is critical for the metastatic ability was released by the addition of SDS–polyacrylamide gel electrophor-
associated with mesenchymal phenotypes of basal-type breast esis sample buffer. The amount of activated KRAS was determined by
cancer cells, suggesting that KRAS signaling is a novel immunoblotting with a KRAS antibody.
therapeutic target for basal-type breast cancer.
Transfection
Small interference RNA (siRNA) duplexes were introduced into cells
MATERIALS AND METHODS
using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA)
Cell culture
according to the procedure recommended by the manufacturer. Cells
Human breast epithelial cell line MCF10A, breast cancer cell lines,
were collected after 48 h for subsequent experiments. All siRNA were
MCF-7, SK-BR3, BT549, BT474, T47D, MDA-MB-231, Hs578t and
purchased from Samchully Pharmaceutical Co. Ltd. (Seoul, Korea).
BT20 were established from the American Type Culture Collection
(Manassas, VA, USA). Cells were cultured in a humidified 5% CO2
Transduction
atmosphere at 37 °C. The normal human breast epithelial cell line
Oncogenic KRAS (G13D) was cloned into retroviral vector MFG. For
MCF10A was maintained in Dulbecco’s Modified Eagle’s/F-12 med-
retrovirus production, H29D cells were cultured in Dulbecco’s
ium supplemented with 5% heat-inactivated horse serum (Invitrogen,
Modified Eagle’s medium (Invitrogen) supplemented with 10% fetal
Seoul, Korea), 10 μg ml − 1 insulin, 20 ng ml − 1 EGF, 0.1 μg ml − 1
bovine serum, 2 mmol l − 1 GlutaMAX (Invitrogen), 50 units per ml
cholera toxin, 0.5 μg ml − 1 hydrocortisone, penicillin (100 units per
ml) and streptomycin (100 μg ml − 1). MCF7 cells were grown in penicillin/streptomycin, 1 μg ml − 1 tetracycline, 2 μg ml − 1 puromycin
minimum Eagle’s medium supplemented with 10% fetal bovine and 0.6 mg ml − 1 G418 sulfate (Calbiochem, San Diego, CA, USA) and
serum, penicillin (100 units per ml) and streptomycin (100 g ml − 1). transfected with MFG or MFG-KRAS using the Lipofectamine 2000
MDA-MB-231, BT474, T47D, Hs578t and SK-BR3 cells were grown in reagent (Invitrogen). Forty-eight hours after the transfection, viral
Dulbecco’s Modified Eagle’s medium; BT549 and BT20 cells in RPMI supernatant was harvested and passed through a 0.45-μm filter, and
supplemented with 10% fetal bovine serum, penicillin (100 units per the viral supernatant was frozen at − 80 °C. The supernatant was used
ml) and streptomycin (100 g ml − 1). for infection after adding 4 μg ml − 1 Polybrene (Sigma).
Figure 1 KRAS activity is critical for mesenchymal phenotypes of basal-type breast cancer cells. (a) Activated KRAS affinity precipitation
assay and western blot for KRAS comparing basal- and luminal-type of breast cancer cell lines. Migration and invasion assay of MDA-
MB231 (b) and BT549 (c) basal-type breast cancer cells after treatment with siRNA targeting KRAS. β-Actin was used as a loading
control. Error bars represent mean ± s.d. of triplicate samples. KRAS, Kirsten rat sarcoma viral oncogene homolog; si-cont, scrambled
control siRNA; si-KRAS, siRNA targeting KRAS. *Po0.01 versus control.
siRNA-mediated downregulation of KRAS effectively attenu- in basal-type breast cancer cells. Notably, SLUG expression was
ated migratory and invasive properties both in MDA-MB231 markedly decreased by downregulation of KRAS, whereas
and BT549 cells (Figures 1b and c). Taken together, these SNAIL, ZEB1 and TWIST expression were not altered,
results suggest that KRAS is preferentially expressed and indicating that KRAS promotes EMT through SLUG in breast
activated in basal-type breast cancer cells and contributes to cancer cells (Figure 2c). Taken together, these results suggest
invasiveness of basal-type breast cancer cells. that activation status of KRAS is critical for the mesenchymal
phenotype and metastatic ability of basal-type breast
KRAS is necessary for the maintenance of mesenchymal cancer cells.
phenotypes of basal-type breast cancer cells
Tumor cell invasion is involved with the loss of cell–cell Ectopic expression of KRAS confers mesenchymal
interaction together with the acquisition of migratory proper- phenotypes on luminal-type breast cancer cells
ties, and is often associated with EMT.13 Very recently, several Since KRAS is necessary for the maintenance of mesenchymal
lines of evidence suggested that expression of EMT markers phenotypes in basal-type breast cancer cells, we next examined
and regulators are closely associated with the basal-type breast whether ectopic expression of KRAS confers mesenchymal
cancer cells.4,5 We next examined whether KRAS promotes phenotypes on luminal-type breast cancer cells. To this end, we
invasiveness of basal-type breast cancer cells through EMT. overexpressed oncogenic mutant form of KRAS G13D in
Notably, siRNA-mediated downregulation of KRAS caused a relatively less malignant luminal-type MCF7 breast cancer cells
decrease in the mesenchymal cell markers such as VIM and (Figure 3a). We next examined whether exogenous KRAS
FN1 in MDA-MB231 cells (Figures 2a and b). In the BT549 cell transduces the downstream signaling pathways of KRAS.
line, downregulation of KRAS caused an increase in the Importantly, retrovirus-mediated delivery of the oncogenic
epithelial cell marker E-cadherin and a decrease in the KRAS enhanced the kinase activity of RAF-1 and increased
mesenchymal cell markers N-cadherin and VIM. We next the phosphorylation of ERK (Figure 3b). Also, oncogenic
examined EMT transcription factors, such as SLUG, SNAIL, KRAS expression increased the activity of PI3K and phosphor-
ZEB1 and TWIST, after treatment with siRNA targeting KRAS ylation of AKT on Thr308 and Ser473 (Figure 3c). Thus,
Figure 2 KRAS is necessary for the maintenance of mesenchymal features of basal-type breast cancer cells. Western blot analysis (a) and
immunocytochemistry (b) for EMT markers such as FN1, VIM, E-cadherin and N-cadherin in basal-type breast cancer cells after treatment
with siRNA targeting KRAS. (c) Western blot analysis for EMT master regulators such as SLUG, SNAIL, ZEB1 and TWIST in basal-type
MDA-MB231 breast cancer cells after treatment with siRNA targeting KRAS. β-Actin was used as a loading control. DAPI, 4, 6-diamidino-
2-phenylindole; KRAS, Kirsten rat sarcoma viral oncogene homolog; si-cont, scrambled control siRNA; si-KRAS, siRNA targeting KRAS;
VIM, vimentin. Error bars represent mean ± s.d. of triplicate samples. *Po0.01 versus control.
Figure 3 Exogenous expression of KRAS in luminal-type breast cancer cells. Western blot analysis for KRAS (a) and its major downstream
effectors, RAF-1/ERK (b) and PI3K/AKT (c) in luminal-type MCF7 breast cancer cells that are transduced with KRAS or control vector
MFG. β-Actin was used as a loading control. KRAS, Kirsten rat sarcoma viral oncogene homolog.
retrovirus-mediated delivery of the oncogenic KRAS caused mesenchymal phenotypes in normal mammary cells similar
activation of RAF-1/ERK and PI3K/AKT signaling pathways, to breast cancer cells.
the major downstream effectors of KRAS. By overexpression of
KRAS, we further found that oncogenic KRAS signaling confers Knockdown of KRAS suppresses in vivo metastatic ability of
migratory and invasive properties onto MCF7 and SKBR3 basal-type breast cancer cells
luminal-type breast cancer cells (Figures 4a and b). In parallel, As the above data showed that KRAS is a critical regulator for
exogenous expression of KRAS caused induction of mesench- mesenchymal phenotypes and metastatic ability of basal-type
ymal markers such as N-cadherin and VIM, while it decreased breast cancer cells, we next evaluated our findings in vivo. To
epithelial marker E-cadherin in luminal-type MCF7 breast this end, MDA-MB231 cells were transfected with siRNA-
cancer cells (Figure 4c). Consistent with this data, expression of targeting KRAS or scrambled control siRNA, and then injected
into the tail veins of athymic nude mice (Figure 6a). By
KRAS in another luminal-type SKBR3 also induced mesench-
5 weeks, the frequency of metastasis was analyzed by counting
ymal markers such as N-cadherin and VIM. Moreover, KRAS
the number of metastatic foci on the surface of whole lungs
expression induced EMT transcription factor SLUG in both
with naked eyes. Of note, lung metastasis was effectively
MCF7 and SKBR3 cells (Figure 4d). Taken together, these
blocked by the downregulation of KRAS in MDA-MB231
results suggest that expression of KRAS alone can cause
breast cancer cells (Figure 6b). Since metastasis is the primary
mesenchymal features on luminal-type breast cancer cells. cause of death in breast cancer patients, we also examined the
correlation of KRAS expression levels with prognosis of breast
Oncogenic KRAS promotes mesenchymal phenotypes in
cancer patients. Using an online survival analysis tool, we
normal mammary cells
analyzed the correlation of KRAS expression levels with
To extend our observations, we also transduced normal
relapse-free survival rate by the Kaplan–Meier plot.14 Impor-
mammary MCF10A cells with the oncogenic mutant form of
tantly, breast cancer patients with higher levels of KRAS
KRAS G13D. In line with the above data, expression of
displayed significantly a lower relapse-free survival rate com-
oncogenic KRAS G13D also increased the migratory and pared with patients having relatively lower levels of KRAS
invasive properties in normal mammary MCF10A cells (Figure 6c). Collectively, these results suggest that KRAS is a
(Figure 5a), accompanying with EMT markers such as E-cad- critical regulator for the metastasis of breast cancer cells, having
herin, N-cadherin and VIM (Figure 5b). Notably, as in a correlation with prognosis of breast cancer patients.
luminal-type breast cancer cells, overexpression of oncogenic
KRAS in MCF10A cells also caused an increase of SLUG, while DISCUSSION
other EMT transcription factors (SNAIL, ZEB1, TWIST) were Basal-type breast cancers are known to be the most aggressive
not altered by KRAS expression (Figure 5c), indicating that and deadly breast cancer subtypes with high rates of tumor
KRAS promotes EMT through upregulation of SLUG. Taken recurrence and poor overall survival.4,15,16 Previous studies
together, these results suggest that KRAS also can induce have shown that expression of EMT markers and regulators are
Figure 4 Exogenous expression of KRAS promotes migration and invasion of luminal-type breast cancer cells through EMT. Wound healing
(a), migration and invasion assays (b) of MCF7 and SKBR3 luminal-type breast cancer cells that are transduced with KRAS or control
vector MFG. Western blot analysis for EMT markers (c) and EMT regulators (d) in KRAS or control vector MFG-transduced MCF7 and
SKBR3 luminal-type breast cancer cells. β-Actin was used as a loading control. Error bars represent mean ± s.d. of triplicate samples.
*Po0.01 versus control. KRAS, Kirsten rat sarcoma viral oncogene homolog; VIM, vimentin.
closely associated with the basal-subtype breast cancer cells, all of those cancer cells carried wild-type KRAS except MDA-
indicating that basal-type breast cancer cells display more MB231 cells. Previously, RAS mutations are found in at least
mesenchymal phenotypes compared with the luminal cancer 20% of all human malignancies, with KRAS being the most
cells.4,5 Because of their mesenchymal features, basal-type frequently activated oncogene.9,10 However, despite the obser-
breast cancer cells appear to be highly invasive and display vations that KRAS is often aberrantly activated in breast
metastatic ability. However, the molecular mechanisms under- cancers,17 the importance of KRAS relatively has not been
lying the maintenance of mesenchymal phenotypes along with intensively focused in breast cancers due to its low frequency of
metastatic ability in basal-type breast cancer cells remain mutation.18 In this study, by using an endogenous or exogen-
obscure. ous KRAS-driven cancer-progression model, we found that
In this current study, we observed that KRAS signaling has a aberrant KRAS activation is critical for mesenchymal pheno-
correlation with characteristics of basal-type breast cancer cells types and metastatic ability of basal-type breast cancer cells.
that are more malignant and metastatic subtype. Importantly, Our in vitro and in vivo studies demonstrated that the down-
both the expression and activity of KRAS were markedly higher regulation of KRAS effectively suppresses mesenchymal pheno-
in basal-type breast cancer cells than the luminal type, although types and metastatic ability of basal-type breast cancer cells.
Figure 5 Exogenous expression of KRAS promotes migration and invasion of normal mammary cells through EMT. (a) Migration and
invasion assays of MCF10A normal mammary cells that are transduced with KRAS or control vector MFG. Western blot analysis for EMT
markers (b) and EMT regulators (c) in KRAS or control vector MFG-transduced MCF10A normal mammary cells. β-Actin was used as a
loading control. Error bars represent mean ± s.d. of triplicate samples. *Po0.01 versus control. KRAS, Kirsten rat sarcoma viral oncogene
homolog; VIM, vimentin.
In contrast, ectopic expression of KRAS conferred mesenchy- selected with adopting the most powerful signaling pathways
mal phenotypes on luminal-type breast cancer cells as well as for survival and proliferation. In this regard, the fact that basal-
normal mammary epithelial cells, suggesting that KRAS type breast cancer cells have higher levels of KRAS, albeit lower
signaling is necessary and sufficient for the acquisition of mutation frequency, implicates that KRAS is a strong onco-
metastatic behavior associated with mesenchymal phenotypes. genic signal that might be adopted by basal-type breast cancer
Supporting our findings, Eckert et al.17 found that RAS is cells, conferring mesenchymal phenotypes on breast
frequently activated in breast carcinoma cells that lack mutated cancer cells.
RAS. In line with this observation, a functional germline In this current study, we found that downregulation of
variant in the 3′-UTR of the KRAS oncogene (rs61764370 KRAS decreases mesenchymal features of basal-type breast
T4G) has been recently identified and was reported to be cancer cells via inhibition of SLUG among EMT transcription
associated with increased risk of both invasive epithelial ovarian factors. Consistently, exogenous expression of KRAS caused an
cancer19 and breast cancer20 in clinically well-annotated increase of SLUG expression, but not SNAIL, TWIST and
cohorts. This functional variant has been shown to disrupt a ZEB1, in luminal-type breast cancer cells. In agreement with
let-7 miRNA binding site leading to increased expression of our finding, previous studies suggested that SLUG is highly
KRAS in vitro. Notably, the frequency of KRAS-variant in expressed in basal-type breast cancer cells and is critical to
breast cancer was found to be associated with basal-type breast determine basal- and luminal-type breast cancer.22–24 Taken
cancer.20,21 In cancer progression, cancer cells are clonally together with the previous studies, our findings suggest that
Figure 6 KRAS has a correlation with in vivo metastatic ability of basal-type breast cancer cells and prognosis of breast cancer patients.
(a) Schematic experimental procedure for tail vein injection of breast cancer cells that are transfected with siRNA targeting KRAS or
scrambled control siRNA into athymic BALB/c nude mice. (b) Representative images of whole-lung metastasis (left) and quantification of
the metastases foci (right) generated by MDA-MB231 cells after tail vein injection (n = 4). (c) Kaplan–Meier survival curves of human
breast cancer patients using publicly available clinical breast cancer database. Breast cancer patients (3455) were grouped to high (2352)
and low expression (1103) of KRAS. Note that KRAS expression level has a correlation with poor survival rate. KRAS, Kirsten rat sarcoma
viral oncogene homolog. si-cont, scrambled control siRNA; si-KRAS, siRNA targeting KRAS. *Po0.01 versus control.
KRAS causes basal-type breast cancer cells to maintain government, through its National Nuclear Technology Program
mesenchymal phenotypes through the regulation of SLUG (NRF-2013M2A2A7066345; NRF-2012M2B2B1055639).
expression.
In summary, KRAS is preferentially activated in basal-type
breast cancer cells, compared with the luminal type. By the loss
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