RAC3 influences the chemoresistance of colon cancer cells through autophagy and apoptosis inhibition
Rubio et al. Cancer Cell Int
RAC3 influences the chemoresistance of colon cancer cells through autophagy and apoptosis inhibition
María Fernanda Rubio 0 2 3
María Cecilia Lira 0 2 3
Francisco Damián Rosa 0 2 3
Adrían Dario Sambresqui 1 3
María Cecilia Salazar Güemes 3
Mónica Alejandra Costas 0 2 3
0 Instituto de Investigaciones Medicas (IDIM) Laboratory of Molecular Biology and Apoptosis, Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad de Buenos Aires , Buenos Aires , Argentina
1 Department of Gastroenterology, Instituto de Investigaciones Médicas Dr. A. Lanari, UBA , Buenos Aires , Argentina
2 Instituto de Investigaciones Medicas (IDIM) Laboratory of Molecular Biology and Apoptosis, Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad de Buenos Aires , Buenos Aires , Argentina
3 Universidad de Buenos Aires, Facultad de Medicina, Instituto de Investigaciones Médicas A Lanari , Buenos Aires , Argentina
Background: RAC3 coactivator overexpression has been implicated in tumorigenesis, contributing to inhibition of apoptosis and autophagy. Both mechanisms are involved in resistance to treatment with chemotherapeutic agents. The aim of this study was to investigate its role in chemoresistance of colorectal cancer. Methods: The sensitivity to 5-fluorouracil and oxaliplatin in colon cancer cells HT-29, HCT 116 and Lovo cell lines, expressing high or low natural levels of RAC3, was investigated using viability assays. Results: In HCT 116 cells, we found that although 5-fluorouracil was a poor inducer of apoptosis, autophagy was strongly induced, while oxaliplatin has shown a similar ability to induce both of them. However, in HCT 116 cells expressing a short hairpin RNA for RAC3, we found an increased sensitivity to both drugs if it is compared with control cells. 5-Fluorouracil and oxaliplatin treatment lead to an enhanced caspase 3-dependent apoptosis and produce an increase of autophagy. In addition, both process have shown to be trigged faster than in control cells, starting earlier after stimulation. Conclusions: Our results suggest that RAC3 expression levels influence the sensitivity to chemotherapeutic drugs. Therefore, the knowledge of RAC3 expression levels in tumoral samples could be an important contribution to design new improved therapeutic strategies in the future.
Colorectal cancer; RAC3; Chemoresistance; Apoptosis; Autophagy
Colorectal cancer (CRC) is one of the most commonly
diagnosed cancers, and 1.23 million patients are
diagnosed with CRC worldwide each year [
]. Despite recent
advances in chemotherapies that have improved survival
rates, patients with late-stage disease and elderly [
have a poor prognosis, and the overall mortality rate of
CRC is approximately 40% [
Although CRC has been widely studied and the role
of cancer stem cells in the persistence and expansion of
this disease shows to be central, the genetic changes and
molecular mechanisms underlying the development and
progression of this cancer are not completely understood
In CRC, chromosomal aberrations have been
extensively analyzed by comparative genomic hybridization,
and several frequently amplified regions, including 20q,
have been identified [
]. Amplification of 20q has been
also detected frequently in many other cancers, like
breast, ovarian, gastric, bladder, and hepatocellular
]. Amplification and overexpression of an
oncogene have been shown to play an important role in
the pathogenesis of various cancers, probably because
overexpression of the amplified oncogene confers a
growth advantage. Several candidate oncogenes have
been isolated from 20q, including AIB1 at 20q12 .
Amplified in breast cancer 1 (AIB1), also known
as Receptor associated-coactivator 3 (RAC3, SRC-3,
ACTR, TRAM2, pCIP and NCoA3), is a member of the
SRC/p160 coactivator family that also includes SRC-1
(NCoA1) and SRC-2 (GRIP1, TIF2 and NCoA2) [
RAC3 is highly expressed in several human cancers
such as breast cancer [
], prostate cancer [
] and liver
] and has been demonstrated to be a key
regulator in tumor initiation, progression, metastasis
and survival [
18, 20, 21
]. RAC3 can interact with nuclear
receptors and other transcription factors to regulate the
expression of their target genes involved in many
signaling pathways, including ERα, E2F1, NF-κB and HER2/
]. It has been reported that RAC3 is
overexpressed in 35% of human CRC samples [
]; however, the
role of RAC3 in CRC progression is still unknown.
We have previously found that RAC3 overexpression
has an anti-apoptotic and anti-autophagic role [
not only through its nuclear action, but also by positively
regulating the activity of p38 and Akt kinases,
inhibiting caspase-8 and -9 and blocking apoptosis-inducing
factor-1 (AIF-1) translocation from mitochondria to the
In this study we investigated the effect of RAC3
expression levels in the sensitivity to several
chemotherapeutic drugs. We found that expression of this oncogene is
significantly higher in some CRC cell lines. Interestingly,
the sensitivity to chemotherapy treatments could be
effectively improved by decreasing RAC3 expression and
increasing the apoptotic and autophagic responses.
Patients and tissue specimens
CRC specimens tissues (n = 14) and normal tissue
(n = 3) (Table 1) were obtained from Instituto de
Investigaciones Médicas Dr. A. Lanari FMED-UBA, Buenos
Aires, Argentina. All samples were confirmed by
pathological examination, and staging was performed
according to the 1997 CRC staging system of the UICC. Among
the 14 CRC samples, 5 were from male patients and 9
were from female patients, with ages ranging from 68
to 91 years (median, 81.1 years). Informed consent was
obtained from all patients, and this study was approved
by the ethics committee of Instituto de Investigaciones
Médicas Dr. A. Lanari.
Paraffin-embedded tissue blocks were sectioned,
deparaffinized in xylene and rehydrated for
immunohistochemical staining [
]. Antigen retrieval was performed
using sodium citrate. The sections were then
incubated in H2O2 (3%) for 10 min, blocked in horse serum
for 60 min and incubated with an anti-RAC3 antibody
(Santa Cruz Biotechology, 1:100) at 4 °C overnight. After
incubation with a universal biotinylated secondary
antibody for 60 min, the specimens were incubated with
H2O2-diaminobenzidine (DAB) until the desired staining
intensity was observed. The sections were counterstained
with hematoxylin, dehydrated and mounted.
Cell culture and reagents
Human CRC cell lines HT-29 (ATCC® HTB-38™), HCT
116 (ATCC® CCL-247™) and LoVo (ATCC® CCL-229™)
were purchased from American Type Culture Collection
(ATCC® Manassas, VA, USA). All cells were maintained
in DMEM/F12 (Invitrogen Corp., USA) supplemented
with 10% fetal bovine serum (FBS) (Invitrogen Corp.,
USA) and cells were incubated at 37 °C in a humidified
incubator containing 5% CO2.
Unless stated, reagents were obtained from Sigma
Chemical Co. (St Louis, MO), Thermo Fisher Scientific
(Waltham, MA) or Santa Cruz Biotechnology, USA.
Plasmid construction and transfection
The short hairpin RNA (shRNA) target sequence for
RAC3 (shRAC3) and scramble (control) were previously
developed in our laboratory and cloned into the HuSH
plasmid (OriGeneTechnologies, USA), shRAC3 sequence:
shRAC3 (2) sequence: CCACATTGCCTCTTCGGTCTA
ATAGCATA (data not shown) [
]. LoVo cells were
transfected with a plasmid expressing full-length cDNA of
RAC3 or with empty vector (control). HCT 116 and LoVo
cells were transfected using Lipofectamine 2000
(Invitrogen Corp., USA) according to the manufacturer’s
protocol. Three days after transfection, the cells were incubated
in selection medium containing 0.5 μg/ml Puromycin
(HCT 116) or 0.5 mg/ml Geneticin (LoVo)
(Invitrogen Corp., USA). After 14 days of selection, protein and
mRNA expression were analyzed by immunoblotting and
quantitative Real Time PCR (qPCR).
In reporter assays, HCT 116 control and shRAC3 cells
were transfected with the reporter plasmid containing
the NF-κB consensus binding sequence (κB-Luc) plus
RSV-β-Gal as it was previously described [
Primers and qPCR
Total RNA was extracted using TRIzol reagent
(Invitrogen Corp., USA) and was employed to
generate cDNA using Superscript III RT (Invitrogen Corp.,
USA) and an oligo(dT) primer. qPCR was performed
using LightCycler® 480 SYBR Green I Master (Roche,
USA) according to the manufacturer’s protocol. For
gene expression analysis, qPCR was performed by
using sequence-specific primers for: RAC3 forward
5′-AAGTGAAGAGGGATCTGGA-3′ and reverse
5′-CAGATGACTACCATTTGAGG-3′, CD39 forward
5′-AGCAGCTGAAATATGCTGGC-3′ and reverse
For all analysis, GAPDH forward 5′-TCTCCTCTGA
CTTCAACAGC-3′ and reverse 5′-GTTGTCATACCAGG
AAATGA-3′ was used as an internal control.
Colorectal cancer cells lines were plated in 96-well flat
bottom plates at a density of 8000 cells/well in 100 μl of
medium. After 24 h, cells were stimulated with
5-fluorouracil (FUra 0–150 μM) or oxaliplatin (Oxa 0–50 μM).
Cells were fixed at specific time points and the cell
viability was determined by staining with 0.5% crystal
violet. Absorbance of surviving stained cells was measured
at 570 nm. The percentage of surviving cells was
determined with respect to basal conditions (without any
Half maximal inhibitory concentration 50 (IC50)
values were calculated with GraphPad Prism software
(GraphPad Software Inc., USA) using the sigmoidal
dose–response function. Assays were carried out in
triplicate and at least three independent experiments
Western blot analysis
HT-29, LoVo wt, control or RAC3, HCT 116 wt,
control and shRAC3 cells were harvested and lysed in RIPA
buffer with protease inhibitors [
]. Then, cell lysates
were separated via 6% SDS-PAGE and transferred to
nitrocellulose membrane. The membranes were blocked
in 10% skim milk and incubated with anti-RAC3 (Santa
Cruz Biotechnology, USA).
For apoptosis experiments, cells were stimulated with
FUra (3.5 μM) or Oxa (0.4 μM) for 6 or 24 h. For
Western blot of LC3II/I, cells were pre-incubated with 10 μg/
ml E64D and pepstatin A lysosomal protease inhibitors,
before incubation with FUra (3.5 μM) or Oxa (0.4 μM)
for 90 min. Then, cells were lysed as described before.
Samples were separated by 10 or 15% SDS-PAGE and
electro-transferred to nitrocellulose membranes.
Membranes were blocked in 10% skim milk and incubated
with anti-pro-Caspase 3, Beclin 1 and LC3 antibodies.
The anti-Tubulin antibody was used as an internal
control (Santa Cruz Biotechnology, USA) in all the assays.
Subsequently, all membranes were incubated for 1 h
with horseradish peroxidase-conjugated secondary
antibody, and the specific bands were visualized by
autoradiography using the chemiluminescence luminol reagent
(Santa Cruz Biotechnology, USA).
Microscopy and immunofluorescence assays
HT-29, LoVo wt, control or RAC3, HCT 116 wt,
control and shRAC3 cells were seeded in 24-well plates on
12 mm glass coverslips. After 24 h, cells were stimulated
with FUra (3.5 μM) or Oxa (0.4 μM) for 1, 6 or 24 h. For
acetylated proteins assays, cells were pre-treated with a
deacetylase inhibitor, Trichostatin A (0.4 μM TSA) and
after 30 min stimulated with 0.4 μM Oxa or 3.5 μM FUra
for 6 or 24 h.
For immunofluorescence assays the cells were fixed
with 3% formaldehyde and 0.02% glutaraldehyde for
15 min. Incubation with primary antibody against
Lysacetylated (Cell Signaling Technology, Danvers, MA,
USA) were performed 1 h room temperature in PBS
with 10% FBS. Then, cells were washed with PBS,
incubated with a TRITC-labeled secondary antibody for 1 h,
washed with PBS, mounted on glass slides with PBS/
Glycerol 1:1 solution.
Some cultures were stained with ethidium bromide
(EtBr) and the morphology of death and surviving cells
was observed by fluorescent microscopy. Ethidium
bromide only enters into non-viable cells and stains
chromatin with dark orange color [
Autophagy induction was monitored by monodancyl
cadaverine (MDC) staining and the percentage of cells
showing an aggregated stain was determined by
counting a minimum of 100 cells per slide using fluorescence
For all the assays cells were analyzed with an Olympus
BX51 fluorescent microscope and 100 cells per field were
counted. Images were taken with a digital camera and
analyzed with NIH-ImageJ software.
Analysis of Gene Expression Omnibus (GEO)
To compare the RAC3 expression levels between
metastatic and primary lesions, in patients sensitive or not to
Folic acid-5-fluorouracil and oxaliplatin treatment
(FOLFOX), we used values obtained from GSE28702 data
bank, platform GPL570 Affymetrix (Santa Clara, CA,
HCT 116 cells were plated in 24-well plates 24 h prior
to transfection at a density of 250,000 cells/well. Cells
were transiently transfected with a total of 0.5 μg of DNA
(including 75 ng of κB-luc and 50 ng RSV-β Gal
vectors) using Lipofectamine 2000 protocol as previously
]. The medium was replaced after 5 and 24 h
later cells were pre-incubated with NF-κB inhibitor
Sulfasalazine (SSZ 0.5 mM) for 30 min before stimulation
with 3.5 μM FUra or 0.4 μM Oxa.
The assays for luciferase and β-galactosidase activity
were performed after 24 h of treatment using the
appropriate substrates in accordance with the manufacturer’s
protocols (Promega corp.). To achieve transfections with
a constant amount of DNA, appropriate amounts of
empty vector (pRC3.1) were added to each well.
At least three independent experiments were carried out
in all cases. Results were expressed as the mean ± SD.
The significance of differences between experimental
conditions was determined using ANOVA and the Tukey
Multiple Comparisons Test for paired observations and
a p < 0.01 was considered statistically significant. For
patients, differences in a given variable between groups
were assessed using Fisher’s exact test and a p < 0.05 was
considered statistically significant.
RAC3 expression levels in CRC patients
Firstly, we investigated the RAC3 expression levels in
patients with colorectal cancer (CRC). Afterwards, the
RAC3 mRNA expression was evaluated by qPCR assays
in 17 exploratory biopsies, 14 of which were confirmed
by pathological examination as CRC and 3 were normal
tissues. RAC3 was found overexpressed in 11 (78%) of
the 14 CRC biopsies and by immunohistochemical
staining it was determined that the expression was mainly
cytoplasmic (Table 1 and Fig. 1a). RAC3 expression was
substantially higher in CRC than in normal colon tissue
samples (p < 0.0294), whereas in studies that included a
greater number of patients (n = 85), overexpression was
observed in 35% of the samples [
]. Although a
correlation between the clinical stage and the overexpression
of RAC3 could be occurring, this hypothesis requires to
be validated in larger cohorts of patients (Table 1 and
Then, we investigated RAC3 expression levels in
metastatic and primary lesions from patients sensitive or not
to FOLFOX (Folic acid-5-fluorouracil and oxaliplatin)
treatment. We analyzed the DataSets records in the Gene
Expression Omnibus (GEO) repository from GSE28702
data bank, platform GPL570 Affymetrix [
]. We found
that patients who do not respond to FOLFOX treatment
have higher coactivator expression levels than those
patients that respond to treatment, being this difference
significantly greater in metastatic lesions (Fig. 1c).
These results suggest that RAC3 could be probably
considered as a predictive marker for CRC chemosensitivity.
(See figure on next page.)
Fig. 1 RAC3 acts as a predictive marker for CRC prognosis and chemotherapeutic response: a representative RAC3 Immunohistochemistry in
normal colorectal and CRC tissues. b The RAC3 expression levels in normal colorectal tissues and CRC tissues at different pathological stages were
determined by qPCR and normalized with GAPDH mRNA. c The RAC3 expression levels are compared between metastatic and primary lesion
in patients sensitive or not to FOLFOX treatment (Folic acid-5-fluorouracil and oxaliplatin). The diagram bars show the average ± S.D. of mRNA
expression log-transformed values from GSE28702 data bank, platform GPL570 Affymetrix (Santa Clara, CA, USA) *p < 0.01 respect to FOLFOX
nonresponder in Metastatic lesion. d RAC3 expression levels in three CRC cell lines are compared with normal colorectal and CRC tissues. The diagram
bars correspond to average ± S.D. of RAC3 mRNA expression obtained by qPCR and normalized to GAPDH mRNA, *p < 0.001 respect to normal
tissue and **p < 0.0001 respect to normal tissue and LoVo cell line. e Western blot was performed to determine the protein levels of RAC3 in the
three CRC cell lines. Relative densitometry units (RDU) correspond to the average of densitometry units respect to Tubulin expression, obtained in
three independent experiments. f, g Cell viability was determined by crystal violet staining and IC50 doses were calculated with GraphPad software.
CRC cells were treated with FUra (0–150 μM) for 72 h (f) or Oxa (0–50 μM) for 24 h (g) p < 0.001 IC50 LoVo respect to HCT 116 to FUra or respect to
HCT116 and HT-29 to Oxa
A re 100
RAC3 expression levels in different CRC cell lines determine
its sensitivity to chemotherapeutic drugs
5-Fluorouracil (FUra) and oxaliplatin (Oxa) are
antimetabolite drugs that are widely used for cancer treatment,
particularly for CRC [
]. Despite the increased
understanding of the mechanism of action of these drugs,
resistance to both of them remains as a significant
limitation for its clinical use.
To explore the potential role of RAC3 in CRC
sensitivity to drugs, we first investigated RAC3 expression
in CRC cell lines (HT-29, HCT 116 and LoVo) by qPCR
and western blot analysis. RAC3 expression levels were
higher in HT-29 and HCT 116 cells than LoVo cells
(Fig. 1d, e). Moreover, the RAC3 levels of this last cell line
were similar to those expressed by normal colon tissues,
as determined by qPCR (Fig. 1d), being not possible to
confirm these results by western blot, due to the limiting
amount of human colon normal tissues.
In view that these CRC cell lines express different levels
of RAC3, they were employed as a model to validate the
results obtained by GEO analysis. Thus, we investigated
the sensitivity to FUra (0–150 μM) and Oxa (0–50 μM).
Cell viability was determined by crystal violet staining
and the IC50 was calculated for each cell type (Fig. 1f, g).
We found that the cell line HT-29 overexpressing RAC3
did not respond to treatment with FUra in the
concentration and time that we used. However, the LoVo cell line,
whose RAC3 expression levels are lower, was more
sensitive to treatment with these drugs (Table 2).
In order to determine whether the sensitivity observed
in the different CRC lines could be dependent of the
RAC3 expression levels, the HCT116 cell line was
transfected with a plasmid containing the shRNA sequence for
RAC3 (shRAC3) that we have previously used in other
published works, having a good efficiency and
] or scramble sequence (control). Concerning the
LoVo cells, that naturally express low RAC3 levels, they
were transfected with aRAC3 expression vector or the
empty vector (control) [
]. The efficiency of knockdown
or overexpression was validated by western blot and
qPCR (Fig. 2a–d).
We found that HCT 116 shRAC3 displayed a
significantly decreased viability in response to
chemotherapeutic drugs comparing to the control (Fig. 2e, g, Table 2).
However, LoVo cells became more resistant to
deathinduced by both chemotherapeutic drugs when RAC3 is
overexpressed (Fig. 2f, h, Table 2). Interestingly, the Oxa
stimulation of this cell line follows a particular
concentration-dependent response whose maximal
proliferation inhibitory effect saturates reaching values that are
below 40% in LoVo wt. However, although RAC3
overexpression significantly decreased the IC50, the curve of
biological response was modified, reaching higher
maximal values of cell death. This unexpected result for Oxa
concentrations higher than 0.4 μM could be probably
explained regarding the anti-senescent and proliferative
stimulating role of RAC3 [
], which perhaps could be
increasing the number of cells capable to respond to Oxa
stimulation. Therefore, taking together all these results
clearly demonstrate that the expression levels of RAC3
may influence the sensitivity to chemotherapeutic drugs,
increasing the chemoresistance when is overexpressed.
The decrease of RAC3 expression levels promotes apoptosis induced by FUra and Oxa
We have previously reported that overexpression of
RAC3 inhibits apoptosis and this is one of the
mechanisms involved in resistance to treatment with
chemotherapeutic agents [
]. Therefore, we decided to
study whether RAC3 would be affecting the sensitivity
to these chemotherapeutics via the inhibition of
apoptosis. HCT 116, LoVo and HT-29 cell lines were stimulated
with a drug concentration close to the IC50 and
regarding the upper and lower limit of the CI95 for each cell line
As shown in Fig. 3a, b and in agreement with that
observed in assays of cell proliferation, HT-29 cells
were not-sensitive to apoptosis induced by both drugs.
Values represent results from at least three independent experiments
% top inhibition (CI95)
Fig. 2 The levels of RAC3 expression affect the response to chemotherapeutic drugs: a–d Knocking down efficiency of shRAC3 in HCT 116 cell
line (a, b) and overexpression of RAC3 in LoVo cells (c, d) were determined by qPCR normalized with GAPDH mRNA (a, c) and Western blot where
RDU correspond to the average of densitometry units respect to the Tubulin expression, obtained in three independent experiments (b, d). e–h
Cell viability was determined by crystal violet staining and IC50 doses were calculated with GraphPad software. CRC cells were treated with FUra
(0–150 μM) for 72 h (e, g) or Oxa (0–50 μM) for 24 h (f, h) p < 0.001 IC50 HCT 116 shRAC3 or LoVo RAC3 respect to HCT 116 or LoVo wt and control
for FUra in HCT 116 and LoVo and Oxa in LoVo cells and p < 0.01 IC50 HCT 116 shRAC3 respect to HCT 116 for Oxa
However, LoVo cells were sensitive to Oxa and
FUrainduced apoptosis after 24 h of treatment, showing a
significant increase in the percentage of cells positive for
Ethidium Bromide (EtBr) (Fig. 3a, b). In the case of HCT
116, only Oxa was able to induce apoptosis after 24 h.
Therefore, using the cells where apoptosis could be
induced, we then investigated if this process could be
affected by different RAC3 expression levels. Thus,
we found that sensitivity to apoptosis of HCT 116 cells
was significantly enhanced when RAC3 was knocked
(shRAC3), showing a significant increase of positive
apoptotic cells after 6 h of treatment, earlier than HCT
116 control cells (Fig. 3c, d). Moreover, they became
sensitive to FUra-induced apoptosis.
In agreement with previous evidences, the active
Caspase 3 is an indicator of late apoptosis, while Beclin 1 is
one of its targets. In addition, the cleavage products of
this last protein inhibit autophagy but promote
]. Then, we analyzed the modulation of these
proteins by drugs stimulation in cells overexpressing or not
RAC3. We found a significant reduction of pro-Caspase
3, accompanied by the increased Beclin 1 cleavage in cells
with low RAC3 expression, after treatment with FUra for
24 h (RDU 1.5 vs 1.1) and Oxa at 6 (1.4 vs 1.2) or 24 h (1.5
vs 1.2), (Fig. 3e).
In order to confirm these results we then investigated
if LoVo cells, which naturally express low levels of RAC3
and are sensitive to apoptosis induced by both drugs, may
be affected by RAC3 overexpression. Therefore, we
analyzed apoptosis in control and RAC3 overexpressing cells
performing the same experiments as in HCT 116 cells.
As shown in Fig. 3f, g, when LoVo cells overexpress RAC3
they became completely resistant to both drugs, at least
in the concentrations that were assayed. As expected, no
cleavage of pro-Caspase 3 and Beclin 1 could be detected
Therefore, our results suggest that expression levels
of RAC3 could be affecting the sensitivity to
apoptosisinduced by chemotherapeutic drugs; however, autophagy
could be also involved.
The increase of autophagy under low RAC3 expression levels maximizes the FUra treatment
Recent studies suggest that autophagy could be playing
an important role in cancer development, determining
the response to anticancer therapy. However, the role
of autophagy in these processes is not at all clear, thus,
depending on the circumstances, it may have
diametrically opposite consequences for the tumor [
Many anticancer agents have been reported to induce
autophagy, supporting the idea that autophagic cell death
may be an important mechanism for tumor cell killing by
these agents [
We have previously demonstrated that RAC3
overexpression inhibits autophagy [
]. Therefore, in order to
investigate whether RAC3 could be affecting autophagy
CRC cell lines, we first investigated if these drugs are
capable to induce this response.
Thus, cell lines were stimulated with FUra (3.5 μM) or
Oxa (0.4 μM) for 1 or 6 h. The Fig. 4a, b show that both
drugs may induce autophagy in all the cell lines,
including HT-29 cells which are resistant to apoptosis. In LoVo
cells, the basal autophagy was naturally high, suggesting
it could be a surviving strategy as previously described
]. In the case of HT29 cells, although both drugs
were unable to induce cell death (Fig. 3b), they trigged
autophagy, suggesting it plays a surviving role, as in LoVo
Being these cells sensitive to autophagy induced by
both drugs, we then analyzed how could be affected by
We found that autophagy started earlier (1 vs 6 h) and
was significantly higher in HCT 116 shRAC3 than in
control HCT 116 (Fig. 4c–e). In the case of LoVo cells,
the RAC3 overexpression significantly inhibited the
autophagy induced by both drugs, as determined by
MDC staining and LC3 detection (Fig. 4f–h).
Hence, in agreement with these results, we found that
FUra was a better inducer of autophagy than apoptosis in
cells that overexpress RAC3, suggesting this is the main
mechanism by which induces cell death. However, the
anti-tumoral effect of Oxa shows to be mainly mediated
The sensitivity to both drugs was significantly increased
under low expression levels of RAC3, showing a greater
apoptosis and autophagy which were evidenced earlier
than in cells overexpressing RAC3.
The oxaliplatin and 5‑fluorouracil—induced decrease of acetylated proteins is enhanced in cells expressing low levels of RAC3
Accumulating evidence suggests that the long-term
success of anti-neoplastic therapies is largely determined
by their capacity to reinstate anticancer
]. One of the requisites of immunogenic
chemotherapy is the induction of autophagy [
]. This process
allows the optimal lysosomal exocytosis of ATP from
dying tumor cells [
] and avoids the up regulation of the
immunosuppressive ecto-ATPase CD39 [
In view that reduction in lysine acetylation of cellular
proteins could be a good inducer of autophagy and ATP
], we investigated the effect of FUra and
Oxa over the degree of protein acetylation. Although
HCT 116 and HT-29 cell lines have similar basal levels
of acetylated proteins, we found that acetylation was
significantly inhibited by drugs stimulation only in HCT116
R.D.U. 1.0 1.1 1.0 1.0 1.3 1.4 R.D.U. 1.0 0.9 1.7 1.0 1.2 1.1
Fig. 4 Autophagy induction by FUra and Oxa is dependent of RAC3 levels: Cells lines were loaded in 24 well plates with slices and after 24 h cells
were stimulated with 3.5 μM FUra or 0.4 μM Oxa. a, c and f Autophagy was determined by staining with monodansylcadaverine (MDC) after 1 or
6 h post-treatment. b, d, g Diagram bars correspond to percentage of MDC positive cells per field (at least 10 fields per sample). Statistical analysis
ANOVA and Tukey post-test n = 3 were performed, b *p < 0.01 3.5 μM FUra at 6 h HT-29 respect to HT-29 basal, **p < 0.001 LoVo basal respect to
HT-29 and HCT 116 basal, LoVo treated at each time respect to LoVo basal, HT-29 0.4 μM Oxa at 6 h respect to HT-29 basal and HCT 116 treated for
6 h with FUra or Oxa respect to HCT 116 basal. d *p < 0.001 3.5 μM FUra and 0.4 μM Oxa at 6 h HCT 116 control respect to HCT 116 control basal,
**p < 0.001 HCT 116 shRAC3 treated respect to shRAC3 basal, ***p < 0.001 shRAC3 respect to control and g *p < 0.01 3.5 μM FUra LoVo control
respect to Lovo control basal and LoVo RAC3 treated with 0.4 μM Oxa for 1 h respect to LoVo RAC3 basal, **p < 0.001 LoVo RAC3 basal respect LoVo
control basal, 3.5 μM FUra at any time LoVo control respect LoVo control basal and 0.4 μM Oxa LoVo RAC3 at 6 h respect to LoVo RAC3 basal. e, h
LC3-II/I levels were determined by Western blot. RDU correspond to the average of densitometry units, respect to Tubulin expression
(Fig. 5a, b), Interestingly, this cell line is which better
respond to autophagy induction (Fig. 4a, b).
Concerning LoVo cells, which naturally express low
levels of RAC3, the basal acetylation was lower than in
the other cell lines and no modulation by drugs
stimulation was observed (Fig. 5a, b).
When we analyzed the effect of RAC3 knocking in
HCT 116, we found that both basal and drug-induced
acetylation at 24 h were significantly inhibited respect to
HCT116 control cells (Fig. 5c, d).
In the case of LoVo cells, RAC3 overexpression induced
a significant increase of basal acetylation that could not
be inhibited by anyone of the drugs (Fig. 5e, f ).
Taken together all these results, we may conclude that
high RAC3 expression contributes to the increased basal
levels of protein acetylation, which could be compatible
with its intrinsic histone acetylase activity [
additional substrates could not be excluded. However,
different levels of RAC3 expression do not modify the
sensitivity to drugs-induced acetylation. Therefore,
despite acetylation inhibition could be involved in
autophagy induction, our results demonstrate that this
is not the mechanism by which RAC3 may modulate the
sensitivity to drugs-induced autophagy.
Despite pre-mortem autophagy is not essential for
chemotherapy-elicited cancer cell death to occur [
it is indispensable for immunogenic cell death (ICD)
through the ATP release into the extracellular space
where it serves as a chemotactic factor to attract
antigenpresenting cells into the neighbor microenvironment of
dying cells [
]. This effect is achieved by the
capacity of extracellular ATP to act on purinergic receptors in
the surface of immature dendritic cell precursors and T
cells . The suppression of autophagy in tumor cells
or CD39 overexpression by tumoral cells, blockade the
capacity of chemotherapy to stimulate the invasion of
tumors by antigen-presenting cells [
Since cells overexpressing RAC3 have a low
druginduced autophagy, we analyzed the CD39 expression
in cells having high and low RAC3 levels. We found that
without stimulation, those cells having low RAC3 (LoVo
control and HCT 116 shRAC3) show a lower
expression of CD39 than cells with high coactivator expression
(HT-29, LoVo RAC3 and HCT 116 control) (Fig. 5g).
Then, we investigated the effect of Oxa and FUra over
HCT 116 and LoVo. We found that Oxa induced a
significant decrease of CD39 in control and shRAC3 HCT116
although not FUra effect was observed in shRAC3
(Fig. 5h). In the case of LoVo cells, both drugs induced
the CD39 inhibition (Fig. 5h).
Therefore, although RAC3 did not affect the
natural biological action of these drugs, cells expressing
low RAC3 were more sensitive to
chemotherapeuticsinduced autophagy and express lower levels of CD39.
Chemoresistance under high RAC3 expression levels could be partially mediated through the enhanced NF‑κB activity
We have previously demonstrated that RAC3 is a NF-κB
] that exerts an anti-apoptotic and
antiautophagic role when is overexpressed, not only
enhancing the expression of NF-κB target genes, but also
through additional cytoplasmic actions [
Moreover, the overexpression of a mutated form of RAC3
lacking the tag nuclear signal, which avoids its nuclear
translocation, also exerts an anti-apoptotic role .
Therefore, in view that RAC3 is mainly localized in
the cytoplasmic compartment of CRC, we investigated
if at least part of the chemoresistance to FUra and
Oxainduced cell death could be mediated through NF-κB
First, we investigated if FUra and Oxa could induce
the NF-κB activation at the concentrations that were
assayed in all the experiments. We found that both
drugs were capable to activate NF-κB in control cells, as
expected for several stress signals or apoptosis inducers
]. This activity was significantly inhibited by the IKK
inhibitor SSZ showing values similar to basal conditions
(Fig. 6a). However, the drugs-induced NF-κB activity was
(See figure on next page.)
Fig. 5 Autophagy induction by FUra and OXA induces a decrease in protein acetylation in HCT 116 cells: a, c and e Cells were pre-treated with a
deacetylase inhibitor TSA (0.4 μM) for 30 min and then, stimulated with FUra (3.5 μM) or Oxa (0.4 μM) for 6 or 24 h. The arrows show the positive
acetylation detected by immunofluorescence using an anti-Lysine acetylated antibody and an antibody anti-mouse coupled to Rodamine. A cell
detail of the 10 × magnification is shown in the square. b, d and f Diagram bars correspond to percentage of acetylated protein per field (at least 10
fields per sample). Statistical analysis ANOVA and Tukey post-test n = 3 were performed. b *p < 0.001 LoVo TSA respect to HT-29 and HCT 116 TSA,
HCT 116 treated respect to HCT 116 TSA, d *p < 0.01 HCT 116 control TSA plus 3.5 μM FUra at 24 h respect to HCT 116 control TSA, **p < 0.001 HCT
116 control TSA plus FUra or Oxa at 6 h respect HCT 116 control TSA and HCT 116 shRAC3 TSA respect HCT 116 control TSA and f *p < 0.001 LoVo
RAC3 with TSA respect to LoVo control TSA. g Expression levels of CD39 in CRC cell lines. The diagram bars show the average ± S.D. of CD39
expression normalized to GAPDH from three independent experiments, *p < 0.001 HCT 116 shRAC3 and LoVo control respect to HCT 116 control and
Lovo RAC3 respect to LoVo control. h The diagram bars show the average ± S.D. of CD39 expression normalized to GAPDH from three independent
experiments, *p < 0.001 HCT 116 shRAC3 basal respect to HCT 116 control basal and **p < 0.01 cells treated with 0.4 μM Oxa respect to HCT 116
control basal or HCT 116 shRAC3 basal
HCT 116 control - SSZ
HCT 116 control +SSZ
HCT 116 shRAC3 - SSZ
HCT 116 shRAC3 +SSZ
basal FUra Oxa
HCT 116 control
basal FUra Oxa
HCT 116 shRAC3
(See figure on previous page.)
Fig. 6 NF-κB activity is partially involved in the chemoresistance induced by high RAC3 expression levels: a HCT 116 control or shRAC3 cells were
co-transfected with κB-Luc reporter plasmid plus RSV-β Gal. Cells were pre-stimulated for 30 min with 0.5 mM SSZ or DMSO, prior to treatment with
3.5 μM FUra or 0.4 μM Oxa for 24 h. The diagram bars correspond to the average ± S.D. of relative light units normalized with the corresponding
β-galactosidase values, *p < 0.01 HCT 116 shRAC3 treated with 3.5 μM FUra respect to HCT 116 shRAC3 basal and ***p < 0.001 HCT 116 control
treated with drugs respect to HCT 116 control basal, HCT 116 control treated with FUra or Oxa plus SSZ respect to HCT 116 control without SSZ and
HCT 116 shRAC3 respect to HCT 116 control. b, c HCT 116 cell lines were pre-stimulated for 30 min with 0.5 mM SSZ and then the cells were treated
for 6 h with 3.5 μM FUra or 0.4 μM Oxa. b Representative microphotography (200×) of apoptotic Ethidium Bromide (EtBr) stained cells after 6 h of
treatment and c Diagram bars correspond to the percentage of EtBr positive cells per field (at least 10 fields per sample), *p < 0.05 HCT 116 control
or shRAC3 FUra plus SSZ respect to HCT 116 treated with FUra and HCT 116 shRAC3 Oxa plus SSZ respect to shRAC3 Oxa, **p < 0.01 HCT 116 control
FUra + SSZ respect to HCT 116 control basal and HCT 116 shRAC3 FUra respect to HCT 116 hsRAC3 basal, ***p < 0.001 HCT 116 control Oxa + SSZ
respect to HCT 116 control basal and Oxa, HCT 116 shRAC3 FUra + SSZ or Oxa + SSZ respect to HCT 116 shRAC3 treated with FUra or Oxa,
significantly lower in shRAC3 than in control cells, as
expected, being RAC3 a required NF-κB coactivator [
Therefore, no effect of SSZ could be detected over
drugsinduced NF-κB activity in shRAC3 cells (Fig. 6a).
Then, using the same SSZ concentration, we
investigated its effect over drugs-induced apoptosis. As shown
in Fig. 6b, c, SSZ significantly increased the FUra and
Oxa-induced apoptosis in both control and shRAC3
HCT 116. Although the increased cell death in control
HCT 116 was almost 100%, a lower effect but still
significant was observed in HCT 116 shRAC3. Therefore,
most of the resistance of RAC3-overexpressing cells to
chemotherapeutics-induced apoptosis could be mainly
mediated through an increased NF-κB activity. However,
our results also suggest that additional signals not related
to this transcription factor but at least dependent of IKK
activity could be involved.
Previous studies demonstrated that RAC3 is a critical
predictor of cancer prognosis in different cancer types
]. The gene for this molecule was reported to be
amplified in 2–10% of human breast tumors and the
protein overexpressed in 30–60% of tumors, suggesting that
RAC3 provides a growth advantage for breast cancer cells
. In CRC studies, a copy number gain of RAC3 was
detected in 27.5% and it is overexpressed in 35% CRC
samples correlating with tumor progression [
Despite all these evidences, the role of RAC3 in CRC
chemoresistance is still unknown.
In the present study, we reported new findings
compelling evidence that RAC3 overexpression in CRC cell lines
decreases the sensitivity to chemotherapeutic drugs and
the mechanism involves the inhibition of apoptosis and
autophagy induced by these agents.
In this work, we found that normal tissues express low
levels of RAC3, but CRC samples shown an
overexpression of this molecule, as expected, while its localization
was mainly cytoplasmic. This last observation could
be particularly interesting concerning the mechanisms
by which RAC3 may contribute to CRC development.
Indeed, it is in agreement with our previous works
demonstrating that besides its nuclear role as a coactivator,
enhancing the NF-κB activity and increasing the
antiapoptotic genes expression, RAC3 has a cytoplasmic
role, regulating kinase activity, interacting with
cytoskeletal proteins, delaying the AIF nuclear translocation and
inhibiting apoptosis [
Concerning the RAC3 anti-autophagic role, we have
previously demonstrated that part of the mechanism
involves NF-κB activation. However, the
overexpression of a truncated form of RAC3 that lacks the signal
for nuclear translocation also exerts an anti-autophagic
role, suggesting that cytoplasmic actions of RAC3 may
contribute to this activity [
]. Therefore, RAC3 is a very
particular molecule which has the ability to modulate
multiple biological responses through several not related
mechanisms in different cellular compartments. Thus,
in the nucleus it can modify the transcriptional
activity of steroid nuclear receptors and transcription factors
through its own histone acetylase activity, but also by
recruitment of other molecules, changing the expression
pattern of a wide amount of target genes [
addition, in the cytoplasm, RAC3 may be associated to
several proteins modulating its activity [
]. Although no
cytoplasmic RAC3 acetylase substrates have been
identified to date, this possibility should not be excluded and
deserves to be investigated, regarding the RAC3
cytoplasmic abundance in some tumors, as well as our own
results concerning the increase of total proteins
acetylation when RAC3 is overexpressed.
Afterwards, the predominantly cytoplasmic
localization observed in CRC tissues could be determinant in
chemotherapeutic response and probably related to the
molecular mechanisms of both apoptosis and autophagy
signaling pathways where RAC3 exerts the inhibitory
effect. However, our results demonstrate that despite its
cytoplasmic localization, most of the anti-apoptotic effect
under chemotherapeutics treatment could be
mediated through an enhanced NF-κB activity, as a nuclear
receptor coactivator. However, additional signals, nuclear
or cytoplasmic, could not be excluded in the
RAC3induced anti-apoptotic and anti-autophagic activity.
In a previous work performed in primary cutaneous
melanoma samples, the authors described RAC3 as a
prognostic marker for the prediction of survival
associated with melanoma [
], while in breast cancer the
RAC3 overexpression was considered as a predictor of
resistance to Tamoxifen treatment [
]. In this regard, in
this work, through the in silico analysis we demonstrate a
correlation between the response to FOLFOX treatment
and the RAC3 expression levels. Therefore, our findings
in CRC are in agreement with other previous works,
supporting that levels of RAC3 expression could have a
possible predictive role concerning chemoresistance.
When we analyzed the expression levels of RAC3 in
patients of the IDIM hospital population and its
correlation with the tumor stage, the results obtained show a
tendency similar to that reported by other authors [
Because our work involves exclusively an aged
population, it is particularly interesting, given that a higher CRC
incidence in patients over 65 has been described in last
recent years [
]. However, preventive care in this
population has only been addressed by a limited number
of guidelines and cancer screening in the elderly (those
greater than 75 years of age) has been controversial [
The lifetime risk of colorectal cancer is ~ 5% with an
incidence in the population over 75 years at about 40–50
per 100,000 persons. However, the incidence falls off to
15–20 per 100,000 in persons 60–65 years [
although we have not performed a comparative study
between young and elderly patients, our findings
contribute to increase the knowledge about molecules to be
studied in CRC, not only as possible predictive
markers of chemoresistance, but also as targets in order to
improve anticancer therapies.
As discussed above, although the inhibitory effects of
RAC3 overexpression on the action of
chemotherapeutic drugs have been investigated, most of these studies
are restricted to its role as a coactivator in the nucleus.
We have previously demonstrated that
RAC3-overexpressing cells are resistant to apoptosis and furthermore,
RAC3-knocked K562 leukemic cell line becomes
sensitive to treatment with flavopiridol [
]. Indeed, in
this work, we demonstrated for the first time that while
RAC3 overexpression inhibits 5-fluorouracil and
oxaliplatin-induced apoptosis, its knocking turns CRC cells into
Several works support a dual role for autophagy in
tumor development: Inducing cell death, or exerting a
surviving role [
]. This last role is in agreement with the
elevated basal autophagy levels usually found in tumoral
cells, like pancreatic cancer [
] as well as the surviving
under stress conditions like hypoxia and starving [
a common situation to which cancer cells are exposed
before the angiogenesis, migration and invasion.
However, it is also known that an exacerbated autophagy
could lead to cell death, and moreover, this could be
the mechanism by which some chemotherapeutic drugs
attack the tumoral cells .
Despite the controversial role of autophagy in cancer
development and tumor progression, recent studies
demonstrate that autophagy has a relevant function in
chemotherapeutic efficiency by induction of ATP exocytosis,
chemotaxis of dendritic cells and ICD induction [
this work, we demonstrate that both 5-fluorouracil and
oxaliplatin are able to induce autophagy more efficiently
in cells that express low levels of RAC3 through at least,
a diminished protein acetylation and CD39 down
regulation. Interestingly, both processes are involved in the
capacity to stimulate the invasion of tumors by
Taken all together these results, we demonstrate for the
first time that RAC3 overexpression could be playing a
critical role in CRC tumoral cells affecting the
sensitivity to apoptosis and autophagy induced by
chemotherapeutic drugs. Moreover, concerning the mechanism, our
results demonstrate that at least part of the
chemoresistance due to RAC3 overexpression could be
dependent of its transcriptional activity as coactivator of NF-κB.
However, in agreement with our results showing a strong
RAC3 cytoplasmic localization in CRC cells, additional
not-nuclear effects could not be excluded, being
perhaps an original and interesting point to be considered
in order to design future improved therapies for CRC
AIB-1: amplified in breast cancer-1; AIF-1: apoptosis-inducing factor-1; CRC:
colorectal cancer; GEO: Gene Expression Onmibus; FOLFOX: folic
acid-5-fluorouracil and oxaliplatin; FUra: 5-fluorouracil; IDC: immunogenic cell death; MDC:
monodansylcadaverine; Oxa: oxaliplatin; RAC3: receptor
associated-coactivator 3; TSA: trichostatin A.
MFR and MAC conceived and designed the study. MCL and FDR performed
the experiments and analyzed the data including IHC. ADS and MCSG
collected clinical specimens. All authors read and approved the final manuscript.
4 Department of Oncology, Instituto de Investigaciones Médicas Dr. A. Lanari,
UBA, Buenos Aires, Argentina.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets analyzed during the current study are available in the GEO
Consent for publication
Ethics approval and consent to participate
All participants provided written informed consent, and the study was
approved by the Institutional Research Ethic Committee of Instituto de
Investigaciones Médicas Dr. A Lanari, Facultad de Medicina, Universidad de
This work has been supported by grants from the National Research Council
of Argentina (CONICET 2015-2017), and Agencia Nacional de Promoción
Científica y Tecnológica, Argentina (ANPCyT Préstamo BID 2014-1424,
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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