Tumor Biol.

DOI 10.1007/s13277-013-1164-6

RESEARCH ARTICLE

High cofilin-1 levels correlate with cisplatin resistance

in lung adenocarcinomas
Matheus Becker & Marco Antônio De Bastiani &
Carolina Beatriz Müller & Melissa M. Markoski &
Mauro Antônio A. Castro & Fábio Klamt

Received: 19 July 2013 / Accepted: 29 August 2013

# International Society of Oncology and BioMarkers (ISOBM) 2013

Abstract High cofilin-1 levels have been shown to be an adenocarcinoma cell panel (P <0.01). In vitro evidences sug-
accurate prognostic biomarker in non-small cell lung gest that cofilin-1 is a biological predictor of cisplatin resis-
cancer (NSCLC) and a predictive factor in drug resis- tance, supporting new treatment initiatives based on cofilin-1
tance. Herein we explore the role of cofilin-1 in cis - levels to guide chemotherapeutic interventions in NSCLC
diamminedichloroplatinum(II) (cisplatin) resistance. We patients.
evaluated cofilin-1 levels in intrinsically cisplatin-
resistant A549 (ICR-A549) cells and determined the cis- Keywords Non-small cell lung cancer . Cisplatin resistance .
platin toxicity in A549 cells transiently transfected and Cofilin-1 . CFL1 . Predictive biomarker
overexpressing CFL1 plasmid. Moreover, expression levels
(activity) of the CFL1 gene network were analyzed in a
cisplatin-resistant human lung adenocarcinoma cell panel. Introduction
ICR-A549 cells, selected by challenging parental cells with
10-fold drug GI50 value, presented a sixfold increase in cis- Non-small cell lung cancer (NSCLC) remains the leading
platin GI50 value and an increased cofilin-1 immunocontent cause of cancer-related mortality worldwide, being responsi-
(P <0.01). In addition, cells transfected with cofilin-1 became ble for almost 1.1 million deaths a year [1]. Unfortunately,
more resistant to cisplatin (P <0.01). High activity of the since most of the cases are diagnosed with advanced patho-
CFL1 gene network was found in a cisplatin-resistant logic (p)-stages of disease, curative pulmonary resection is no
longer a therapeutic option and multimodality treatment be-
came the indicative management of disease [2]. However, the
Matheus Becker and Marco Antônio De Bastiani contributed equally to
this work. effect of current therapies in improving the survival of
NSCLC patients remains far from satisfactory, reflecting that
M. Becker : M. A. De Bastiani : C. B. Müller : F. Klamt (*)
the prognosis of NSCLC is still poor, with a 5-year survival
Laboratório de Bioquímica Celular, Departamento de Bioquímica,
ICBS/UFRGS, 2600 Ramiro Barcelos St, 90035-003 Porto Alegre, probability of 49 % for early stages and less than 1 % for
Rio Grande do Sul, Brazil advanced stages [3].
e-mail: [email protected] Advances in molecular pathology led to the development
M. Becker : M. A. De Bastiani : C. B. Müller : M. A. A. Castro :
of an impressive number of biomarkers that could provide
F. Klamt information about cancer heterogeneity and could have im-
Instituto Nacional de Ciência e Tecnologia-Translacional em portant applications such as prediction and planning of treat-
Medicina (INCT-TM), 90035-903 Porto Alegre, ment [4]. For example, the treatment of NSCLC had been
Rio Grande do Sul, Brazil
revolutionized by the development of targeted agents (e.g.,
M. M. Markoski the FDA-approved drugs erlotinib and gefitinib for pa-
Laboratório de Cardiologia Celular e Molecular, IC/FUC, tients harboring specific EGFR mutations) [5], but deci-
90620-000 Porto Alegre, Rio Grande do Sul, Brazil sion in NSCLC management is still mainly based on the
anatomic extent of the disease. Other factors, such as the
M. A. A. Castro
Hospital de Clínicas de Porto Alegre (HCPA)/UFRGS, molecular characterization of the tumor, are rarely included
90035-903 Porto Alegre, Rio Grande do Sul, Brazil in decision-driven therapeutics [6].
 Tumor Biol.

Despite the large number of studies involving biomarkers room temperature. Bound dye was solubilized with 10 mM
for NSCLC, poor individual performance precludes their in- Tris buffer (pH 10.5), and a plate reader (Spectra Max GEM-
clusion in the clinical practice [7]. Then, the identification of INI XPS, Molecular Devices, USA) was used to measure the
biomarkers that could add value to the TNM system is an optical densities of SRB at 490 nm. Once the cisplatin GI50
important step in an individualized therapy and, ultimately, value was obtained, sub-confluent A549 cells plated in 75-
improves patient survival. cm2 flasks were treated with 10-fold GI50 value for 24 h. The
In this context, we have previously established the role of ICR-A549 cells were left to grow until semi-confluence,
cofilin-1 as a prognostic biomarker for NSCLC patients [8, 9]. harvested, sub-cultured to re-evaluate the cisplatin GI50 value
Using three independent clinical cohorts, we found that as previously described, or collected for Western blot immu-
cofilin-1 levels are highly sensitive and specific in discrimi- noassay. We used rabbit cofilin-1 polyclonal antibody
nating between good and bad NSCLC patient outcomes, (Abcam; 1:2,000) in combination with horseradish
especially in the early disease stage [8, 9]. In these studies, peroxidase-linked secondary antibodies (1:10,000) from
we also found an association between cofilin-1 and lung DakoCytomation. Bands were visualized by chemilumines-
tumor migration and invasion. cence (PIERCE) using X-ray film. Quantification was with
Cofilin-1 (CFL1 gene product; non-muscle isoform; 1072 ImageJ software. Data analyses were performed using
Gene ID) is one of the major proteins responsible for cell GraphPad Prism 5.0 software.
migration processes, playing a key role in actin filament
dynamics [10], and apoptosis induced by oxidants [11]. Transient transfection and overexpression of wild-type
Cofilin-1 is overexpressed in several highly invasive cancer cofilin-1 or mock
cell lines [12–14], as well as in biopsies of oral, renal, and
ovarian carcinomas [15]. More importantly, cofilin-1 levels Transient transfections were performed with Lipofectamine
(protein and mRNA) were found to be correlated with resis- 2000 (Invitrogen) in accordance with the manufacturer's in-
tance to 22 of 33 alkylating drugs tested [8]. These findings structions. Briefly, A549 cells were seeded in 96-well plates
led us to propose the use of cofilin-1 levels as a prognostic and overnight before transfection with 0.2 μg of cofilin-1 plasmid
predictive NSCLC biomarker. (pCMV-XL5) or empty plasmid (mock). DNA was mixed
Herein we aimed to strengthen the association of cofilin- with the liposome reagent at a ratio of 1:2 before addition to
1 with cisplatin resistance in human NSCLC, based on three cells. At 6 h after transfection, the medium was removed and
different experimental strategies: (1) evaluation of cofilin-1 fresh medium was added. Transfection efficiency was de-
immunocontent in the intrinsically cisplatin-resistant A549 termined using a pGFP-N1 vector (Clontech) and evaluat-
(ICR-A549) NSCLC cell line, (2) determination of cisplatin ed by flow cytometry to be ~80 % after 48 h. Cofilin-1
toxicity in A549 cells transiently transfected and overex- levels in transfected cells were determined by dot blot
pressing CFL1 plasmid, and (3) evaluation of the differen- immunoassay, where serial dilutions of samples (1, 2, 4,
tial gene expression level (activity) of the cofilin-1 gene and 8 μL) were applied to a nitrocellulose membrane and
network in response to acute cisplatin treatment and in cofilin-1 immunocontent were determined as described for
cisplatin-resistant human NSCLC cell panel. Western blot.

Differential gene expression and enrichment analysis

Materials and methods
We analyzed differential gene expression levels of the CFL1
Cell line maintenance, treatments, and cisplatin resistance (human cofilin-1) gene network, as previously described [8],
protocol using microarray data from the GSE4127 dataset available at
the Gene Expression Omnibus repository (http://www.ncbi.
Exponentially growing human A549 NSCLC adenocarcino- nlm.nih.gov/geo/). The GSE4127 dataset provides the
ma cells (obtained from NCI-Frederick cell line repository) transcriptional profiling of a set of 10 human lung
were maintained in RPMI 1640 medium (Invitrogen) contain- adenocarcinomas (RERF LC-KJ, ABC-1, PC14, LU65,
ing 10 % fetal bovine serum, 1 μg/mL of amphotericin B, and PC9, PC7, A549, LC2/ad, RERF LC-MS, and PC3 cells),
50 μg/L of garamycin at 37 °C in a humidified atmosphere of with cytotoxicity data of several chemotherapeutic drugs,
5 % CO2. Cisplatin cytotoxicity (GI50 value) was determined including cisplatin and carboplatin [16]. Differential gene
with the sulforhodamine B (SRB) assay as a dose–response expression (activity) and enrichment analysis were obtained
curve, following the NCI-60 drug screening protocol. Briefly, using ViaComplex software version 1.0 [17], which estimates
cells were seeded in a 96-well plate and treated for 72 h after the relative expression level of groups of functionally associ-
overnight adherence. Cells were fixed with 10 % TCA, ated genes (GFAG). Briefly, to obtain a quantitative parameter
washed, and stained with 0.2 % SRB in 1 % acetic acid at that characterizes the functional state of GFAG in the sample,
Tumor Biol.

ViaComplex measures the information content using Bootstrap analysis showed a significant increase in gene ex-
Shannon's entropy. pression level (activity) of the CFL1 gene network in
cisplatin-resistant adenocarcinomas (P <0.01; Fig. 1a). To do
Statistical analysis so, the human lung adenocarcinoma cell lines were first clus-
tered on the basis of cisplatin cytotoxic activity. LC2/ad,
Data are expressed as means±standard deviation of at least RERF-LC-MS, and PC-3 were considered cisplatin-resistant
three independent experiments performed in triplicate. Data cells while RERF-LC-KJ, ABC-1, and PC14 the cisplatin-
were analyzed for significance by Student's t test or by one- sensitive cells (means of the cisplatin GI50 value for each
way ANOVA, with Tukey's multiple comparison post hoc test. group were 12.10±7.97 vs. 2.45±0.43 μM, respectively, P <
Differences were considered statistically significant when P < 0.01; Fig. 1b).
0.05 (GraphPad® Software Inc., 5.0, San Diego, CA, USA). Moreover, we then explored the cofilin-1 immunocontent in
ICR-A549 human adenocarcinoma cells. ICR-A549 cells were
selected by challenging parental A549 cells with 10-fold the
Results GI50 value of cisplatin for 24 h. Approximately 2 weeks after
treatment, the GI50 value for cisplatin was found to be sixfold
In a previous work, exploring data from the NCI-60 cell panel, higher in ICR-A549 cells as compared to parental A549 cells
we found a strong correlation between cofilin-1 (protein and (20.81±8.70 vs. 3.50±0.86 μM, respectively, P <0.0001;
mRNA levels) and increased GI50 value for several clinically Fig. 2a), and ICR-A549 cells presented a significant higher
relevant alkylating agents (including cisplatin and cofilin-1 immunocontent (P <0.01; Fig. 2b). More interesting-
carboplatin). These findings allowed us to propose that ly, A549 cells transiently transfected and overexpressing a
cofilin-1 could be used as a new biological predictor of plasmid containing CFL1 (human cofilin-1 gene) exhibit an
response to this class of anticancer drugs [8]. increase in cisplatin resistance (increase in drug GI50 value), as
Trying to strengthen this observation, we first evaluated the compared to the mock (empty plasmid) group (P <0.01;
expression levels of the CFL1 gene network in an alternative Fig. 3). Representative images of the dot blot immunoassay
cisplatin-resistant human NSCLC cell panel (GSE4127 confirmed the transfection efficacy and showed a significant
dataset). The CFL1 gene network consists of 19 genes increase in cofilin-1 immunocontent after 48 h (P <0.05).
(LIMK1, LIMK2, YWHAG, YWHAZ , TPI1, HSPH1, NRK, All in all, the cumulative experimental data obtained with
ATP1A1, ACTA1, ACTA2, ACTB, ACTC1, ACTG1, TESK1, these in vitro studies support that a high cofilin-1 level is
TESK2 , SSH1 , SSH2 , SSH3 , CAP1 ) identified by the correlated with an increased resistance to cisplatin in human
network-based model of CFL1 interaction partners [8]. NSCLC cell lines.

a b
20 (P < 0.01)
**
Cisplatin GI50 (µM)

15

10

0
lls

lls
ce

ce
ve

nt
ta
iti

is
ns

s
se

re

Experimental Groups
Fig. 1 Differential gene expression levels of the CFL1 gene network in associations according to experimental data (http://string.embl.de/) as
cisplatin-resistant lung adenocarcinoma cell panel. a STRING gene described in Castro et al. [8]. The network drawn was built using a
interactions representation of the CFL1 gene network. A graphic model spring model algorithm. Further details are given in the “Materials and
represents the CFL1 functional gene network vs. cisplatin drug resistance methods” section. This network is significantly enriched with up-
profiles. Gene expression data of cisplatin-resistant cells were crossed regulated genes and compared to a bootstrap null distribution estimated
against cisplatin-sensitive cells. White nodes are genes up-regulated in in the software ViaComplex (P <0.01). b Cisplatin-resistant cells were
resistant phenotype, and black nodes are genes down-regulated in resis- selected as described in the “Materials and methods” section. **P <0.01
tant phenotype (gray nodes are genes not represented in the microarray (different from respective control group; Student's t test)
platform). Connecting lines indicate physical and/or functional
 Tumor Biol.

Fig. 2 Increased cofilin-1 a b

immunocontent in intrinsically A549 cells
1.0 (P < 0.0001)

(relative to untreated cells)

Cisplatin GI50 (fold increase)

cisplatin-resistant A549 (ICR- ICR-A549 cells 8
A549) NSCLC cells. ICR-A549 ***

(relative to parental cells)

Cofilin-1 Immunocontent
(P < 0.01)
cells were selected as described in 15

Cell Survival
6
the “Materials and methods”
section and presented a sixfold **
increase in drug GI50 value (a) GI50 4 10
and an increase in cofilin-1
immunocontent (b) as compared 5
2
to parental A549 cells. Data
represent mean±S.D. of at least
three independent experiments 0.0 0 0
(n =3) performed in triplicate. 0 10 20 30

ls

ls

lls

ls
l

l
ce

ce

l
ce

ce
**P <0.01 (different from Cisplatin (µM)

9
54

54

54

54
respective control group); ***P <

-A

-A
R
0.0001 (Student's t test)

R
IC

IC
19 kDa

Discussion oxidant-induced apoptosis [11]. More importantly, cofilin-1

has been correlated with multidrug resistance in pancreatic
Most of the NSCLC patients are diagnosed at advanced cancers [12] and yeast [25] and with platinum resistance in
stage of disease, and some of them are refractory to ovarian cancer cells [26] and in human lung adenocarcinoma
platinum-based chemotherapy [18]. The primary cause of cell lines and tumor biopsies [27]. These results are in agree-
cancer treatment failure can be found in the biological ment with the findings presented here. Our data also support
properties of the malignant system. In that way, the re- further clinical studies to validate the use of cofilin-1 protein
sponsibility of current chemotherapy inefficiency can be as a new predictive biomarker in non-small cell lung cancer,
directly linked to cancer phenotype [19]. being able to direct decisions in the management of patients
Several studies have consistently correlated cofilin-1 levels with this disease. Therefore, patients with high cofilin-1
with a more aggressive phenotype in different tumor tissues immunocontent may not respond adequately for a chemother-
[8, 20–22]. These observations were attributed to the critical apy treatment based on alkylating agents.
role played by cofilin-1 in the regulation of cellular migration Cisplatin constitutes the major therapeutic option in some
and invasion capacity [15, 23, 24]. In recent years, however, clinical settings and often leads to an initial therapeutic suc-
other functions have been attributed to cofilin-1, such as cess. Still, many patients (in particular, in the context of

a b
8 (P < 0.05) 25 (P < 0.01)
(relative to untransfected)
Cofilin-1 Immunocontent

Cofilin-1 **
Cisplatin GI50 (µM)

Immunocontent 20
6
untransfected
15
mock 4
*
10
24h
2
48h - CFL1 5
72h
0 0
m d

h
h
h
k

1
oc

FL
oc
te

24
48
72
ec

C
sf
an

CFL1 Experimental Groups

tr
un

Experimental Groups

Fig. 3 Transient transfection and overexpression of cofilin-1 lead to an with CFL1 plasmid or mock, A549 cells were treated with different
increase in cisplatin resistance in A549 cells. a Cells were transiently concentrations of cisplatin and drug GI50 were determined. Data represent
transfected with the plasmid containing the cDNA sequence of cofilin-1 mean±S.D. of at least three independent experiments (n =3) performed in
(CFL1) or empty plasmid (mock) as described in the “Materials and triplicate. *P <0.05 (different from respective control group; ANOVA,
methods” section, and cofilin-1 immunocontent was determined by dot using Tukey's multiple comparison post hoc test); **P <0.01 (Student's t
blot analysis in different incubation times. b At 48 h after transfection test)
Tumor Biol.

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Acknowledgments This work was supported by the Brazilian funds
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MCT/CNPq Universal (470306/2011-4), PRONEX/FAPERGS
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