Taxol-resistant breast cancer cells have increased aerobic glycolysis and are more susceptible to

23 Pages·2010·0.78 MB·English

JBC Papers in Press. Published on May 11, 2010 as Manuscript M109.083337
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M109.083337
MICRORNA-125B CONFERS THE RESISTANCE OF BREAST CANCER CELLS TO
PACLITAXEL THROUGH SUPRESSION OF PRO-APOPTOTIC BCL-2 ANTAGONIST
KILLER 1 ( BAK 1) EXPRESSION
Ming Zhou1,3,*, Zixing Liu1,*, Yuhua Zhao1 , Yan Ding1, Hao Liu1, Yaguang Xi1, Wei Xiong3,
Guiyuan Li3, Jianrong Lu5, Oystein Fodstad1,4, Adam I. Riker6 and Ming Tan1,2
1Mitchell Cancer Institute and, 2Department of Cell Biology and Neuroscience, University of South
Alabama, Mobile, Alabama; 3Cancer Research Institute, Central South University, Changsha, China;
4Institute for Cancer Research, The Norwegian Radium Hospital, Oslo, Norway; 5Department of
Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida; 6Ochsner Cancer
Institute, Ochsner Health System, New Orleans, Louisiana
Running title: miR-125b is involved in Paclitaxel resistance

* These authors contributed equally to this work.
Address correspondence to: Ming Tan, 1660 Spring Hill Ave, Mobile, AL 36604. Phone: [phone];
D
FAX: [phone]; E-mail: [email protected] o
w
n
lo
a
d
e
Paclitaxel (Taxol) is an effective demonstrated that the pro-apoptotic Bcl-2 d
fro
chemotherapeutic agent for treatment of cancer antagonist killer 1 (Bak1) is a direct target of m
h
patients. Despite impressive initial clinical miR-125b. Down-regulation of Bak1 suppressed ttp
://w
responses, the majority of patients eventually Taxol-induced apoptosis and led to an increased w
w
develop some degree of resistance to resistance to Taxol. Restoring Bak1 expression .jb
c
.o
Taxol-based therapy. The mechanisms by either miR-125b inhibitor or re-expression of rg
b/
underlying cancer cells resistance to Taxol are Bak1 in miR-125b overexpressing cells y g
u
not fully understood. MicroRNA (miRNA) has recovered Taxol sensitivity, overcoming es
t o
n
emerged to play important roles in miR-125-mediated Taxol resistance. Taken O
c
tumorigenesis and drug resistance. However, together, our data strongly supports a central tob
e
the interaction between the development of role for miR-125b in conferring Taxol r 5
, 2
0
Taxol resistance and miRNA has not been resistance through the suppression of Bak1 1
7
previously explored. In this study, we utilized a expression. This finding has important
miRNA array to compare the differentially implications in the development of targeted
expressed miRNAs in Taxol-resistant and their therapeutics for overcoming Taxol resistance in
Taxol-sensitive parental cells. We verified that a number of different tumor histologies.
miR-125b, miR-221, miR-222, and miR-923
were up-regulated in Taxol-resistant cancer INTRODUCTION
cells by real-time PCR. We further investigated
the role and mechanisms of miR-125b in Taxol Breast cancer is the most common cancer and the
resistance. We found that miR-125b was second leading cause of cancer-related deaths in
up-regulated in Taxol-resistant cells, causing a American women. Paclitaxel (Taxol) has recently
marked inhibition of Taxol-induced cytotoxicity emerged as an important chemotherapeutic agent
and apoptosis, and a subsequent increase in the in the treatment of breast cancer, as well as other
resistance to Taxol in cancer cells. Moreover, we tumor histologies, such as ovarian, prostate, and
1
Copyright 2010 by The American Society for Biochemistry and Molecular Biology, Inc.
non-small cell lung cancers (1, 2). The resistance We aimed to examine the role of miRNAs in
of cancer cells to Taxol and other Taxol resistance in breast cancer cells through the
chemotherapeutic agents frequently results in the screening of differentially expressed miRNAs
subsequent recurrence and metastasis of cancer (3). between Taxol-resistant and Taxol-sensitive cancer
One known mechanism involved with cancer cell cells. We found that miR-125b is up-regulated in
resistance to Taxol is the high expression of the Taxol-resistant cancer cells, with its over-
membrane P-glycoprotein that functions as a expression inhibiting Taxol-induced cytotoxicity
drug-efflux pump (4). Other mechanisms include the and apoptosis, subsequently increasing the
alterations of tubulin structure (5, 6), changes in the resistance of cancer cells to Taxol. Moreover, we
drug-binding affinity of the microtubules (7), and demonstrate that Bak1 is a direct target of
cell cycle deregulation (8, 9). However, the detailed miR-125b in breast cancer cells. Restoring the
molecular mechanisms that may contribute to Taxol expression of Bak1 recovered Taxol-sensitivity in
resistance of cancer cells are still not fully counteracting miR-125b mediated Taxol resistance.
understood. Taken together, miR-125b plays a critical role in
D
microRNA (miRNA) is the non-coding, Taxol resistance through suppression of Bak1 o
w
n
single-stranded RNA of approximately 22 expression and it may serve as a potential target lo
a
d
e
nucleotides, which regulates gene expression and for overcoming Taxol resistance in human breast d
fro
constitutes a novel class of gene regulators (10). cancer. m
h
Mature miRNA molecules are partially ttp
://w
complementary to one or more messenger RNA EXPERIMENTAL PROCEDURES w
w
(mRNA) molecules, and translationally down- .jb
c
.o
regulate gene expression (11, 12) or induce the Cells and cell culture- Human breast cancer cell rg
b/
degradation of mRNAs bearing fully lines, MDA-MB-435 (MDA-435), MDA-MB-231 y
g
u
complementary target sites (13). Among hundreds (MDA-231), MDA-MB-436 (MDA-436), MCF7, es
t o
n
of miRNAs, some of them have been shown to SKBr3 and BT474 were purchased from American O
c
play a role in a variety of biological processes Type Culture Collection (ATCC). Taxol-resistant tob
e
including proliferation, differentiation, migration, 435TR1 (single clone) and 435TRP (pooled clones) r 5
, 2
0
cell cycle, and apoptosis (14, 15). In recent years, cells were developed from MDA-435 cells by 1
7
some miRNAs have been reported to be involved treatment with gradually increasing concentrations
in drug-resistance, acting as potential oncogenes or of Taxol in cell culture medium. Cell viability
tumor suppressors (16-18). The “knockdown” and assay showed that 435TR1 and 435TRP cells
inhibition of miR-221 or miR-222 expression has could tolerate much higher concentrations of Taxol
been shown to sensitize MDA-MB-468 cells to compared to MDA-435 cells, with their IC50
tamoxifen-induced cell growth arrest and concentrations found to be more than 30-fold
apoptosis (17). Ectopic miR-34a expression higher than those of parental MDA-435 cells (19).
resulted in cell cycle arrest and growth inhibition Two more Taxol-resistant cell variants, 436TRP
and attenuated chemoresistance to the anti-cancer and SKBr3-TRP (both are pooled clones), were
drug, camptothecin, by inducing apoptosis (18). developed from MDA-436 and SKBr3 cell lines
To date, however, the interaction between respectively using the same methods as for
miRNAs and Taxol resistance in cancer cells has 435TRP. The cells were cultured in DMEM/F-12
not been explored. (Mediatech Inc.) supplemented with 10% FBS and
2
Penicillin/Streptomycin. by using TaqMan® microRNA reverse
miRNA microarray expression analysis- Total transcription kit (Applied Biosystems) and
RNA was extracted from MDA-435, 435TR1 and TaqMan® microRNA assays kit (Applied
435TRP cells using TRIzol reagent (Invitrogen), Biosystems) by following the manufacturer’s
and the total RNA was labeled with Hy3 and Hy5 protocols. RNU6B served as an internal control.
fluorescent dyes. Pairs of labeled samples were End-point PCR was performed to analyze the
hybridized to miRCURY LNATM microRNA array expression of miR-125b by using a mirVana
slides with coverage of 428 unique human qRT-PCR miRNA Detection Kit and mirVana
microRNAs (Exiqon, 208201-A) by using qRT-PCR Primer Sets (Applied Biosystems)
miRCURY LNATM Power labeling kit and according to the manufacturer’s protocols. Human
miRCURY LNATM microRNA array kit (Exiqon) U6 served as an internal control. For the
according to the manufacturer’s protocol. A expression of Bak1 and GAPDH, cDNAs were
miRNA detection signal threshold was defined as synthesized from 1ug total RNA using Cloned
twice the maximum background signal. AMV First-Strand cDNA Synthesis kit
D
Normalization was done using a cyclic LOWESS (Invitrogen). For quantitative PCR, cDNA was o
w
n
(locally weighted regression) method to remove mixed with 2 × SYBR Green PCR Master Mix lo
a
d
e
the system-related variations. Data adjustments (Applied Biosystems) and various sets of d
fro
included data filtering, log transformation, and gene-specific primers and then subjected to m
2 h
gene centering and normalization. The student t RT-PCR quantification by using the iQ5 real time ttp
://w
test analysis was conducted between MDA-435 PCR system (Bio-Rad). The sequences of the w
w
and 435TR1, MDA-435 and 435TRP samples, and primers used were as follows: Bak1, 5’ primer, .jb
c
.o
miRNAs with P-values < 0.05 were selected for 5'-GCTCCCAACCCATTCACTAC-3', and 3’ rg
b/
cluster analysis. The clustering analysis was done primer, 5’-TCCCTACTCCTTTTCCCTGA-3’; y
g
u
using a hierarchical method and average linkage GAPDH, 5’ primer, 5’-ATCCCATCACCATCTTC es
t o
n
and Euclidean distance metrics. CAG-3’, and 3’ primer, 5’-ATGAGTCCTTCC O
c
Pre-miRNA or anti-miRNA Transfection- miRNA ACGATACC-3’. For semi-quantitative PCR, the tob
e
precursors (pre-miRNAs) and miRNAs antisenses cDNA was mixed with PCR SuperMix (Invitrogen) r 5
, 2
0
(anti-miRNAs) were purchased from Applied and gene-specific primers. The PCR conditions 1
7
Biosystems. Pre-miR-Negative and anti-miR- were 25-30 cycles at 95 °C for 30s, 56 °C for 30s
Negative were used as negative controls. and 72 °C for 1 min. The PCR products were
Oligofectamine Transfection reagent or separated on a 2% agarose gel. All reactions were
Lipofectamine 2000 (Invitrogen) was used for the performed in triplicate. The relative amounts of
transfection of pre-miRNAs or anti-miRNAs. mRNA were calculated by using the comparative
Forty-eight hrs after transfection, the expression of C method. The results are presented as
T
miR-125b was detected by real-time PCR, and the fold-change of each miRNA or mRNA in the
expression of Bak1, a target of miR-125b, was 435TR1 and 435TRP cells relative to the parental
tested by real-time PCR and / or Western blotting. MDA-435 cells.
Quantitative real time PCR (qRT-PCR) and siRNA Experiments- siRNA oligonucleotides for
RT-PCR- Total RNA was isolated from cultured Bak1 were purchased from Sigma, with a
cells using the TRIzol reagent (Invitrogen). For scrambled siRNA (Sigma) used as a control.
miRNA expression analysis, qRT-PCR was done Transfection was performed using the
3
Oligofectamine Transfection reagent (Invitrogen) plates were cotransfected with pMIR-REPORT
according to the manufacturer’s protocol. luciferase reporters with or without 3’-UTR of
Forty-eight hrs after transfection, the cell lysates Bak1, pre-miR-125b or pre-miR-Negative using
were prepared for further analysis by Western Lipofectame 2000 reagent. Twenty-four hrs later,
blotting. cells were harvested and lysed with passive lysis
mRNA Stability- MDA-435 cells were buffer (Promega). Luciferase activity was
transfected with 100 nM pre-miR-125b or measured by using a dual luciferase reporter assay
pre-miR-Negative. After 12 hrs， cells were (Promega). The pRL-TK vector (Promega) was
treated with Actinomycin (5 ug/ml). The stability used as an internal control. The results were
of endogenous Bak1 mRNA was examined with expressed as relative luciferase activity (Firefly
real-time RT-PCR by measuring Bak1 mRNA Luc/Renilla Luc).
levels at the indicated time. Western blotting- Cells were harvested and lysed
Activity of Caspase 3- MDA-435 cells were in a buffer containing 50 mM Tris-HCl, pH 7.5,
transfected with 100 nM pre-miR-125b or 150 mM NaCl, 2 mM EDTA, 1% Triton, 1 mM
D
pre-miR-Negative and seeded into 96-well plates PMSF and Protease Inhibitor Cocktail (Sigma) for o
w
n
at 8 × 103 cells per well in triplicates. After 8 hrs, 20 min on ice. Lysates were cleared by lo
a
d
cells were treated with or without 20 nM Taxol for centrifugation at 14,000 rpm at 4℃ for 10 min. ed
fro
24 hrs. Caspase 3 activity was measured by Supernatants were collected and protein m
h
Caspase-Glo® 3/7 assay kit (Promega) according concentrations were determined by the Bradford ttp
://w
to the manufacturer's instruction. assay (Bio-rad). The proteins were then separated w
w
Apoptosis assay- Cells were transfected with 100 with a SDS/polyacrylamide gel and transferred to .jb
c
.o
nM miR-125b precursor (pre-miR-125b), 100 nM a Nitrocellulose membrane (Bio-rad). After rg
b/
pre-miR-125b in combination with 100 nM blocking in PBS with 5% non-fat dry milk for 1 h, y
g
u
miR-125b inhibitor (anti-miR-125b) or 4 μg Bak1 the membranes were incubated overnight at 4-8℃ es
t o
n
expressing vector respectively, and 24 hrs after with the primary antibodies in PBS with 5% O
c
transfection, the cells were followed with indicated non-fat dry milk. The following antibodies were tob
e
concentrations of Taxol treatment for 48 hrs, and utilized: anti-Bak1 monoclonal antibody (1:1000, r 5
, 2
0
then were subjected to an apoptosis assay. Cell Signaling); anti-cleaved PARP monoclonal 1
7
Apoptosis was determined by Annexin Ⅴ/ antibody (1:1000, Cell Signaling), and anti-β-actin
propidium isodide staining with the Apoptosis monoclonal antibody (1:2000, Sigma). Membranes
Detection Kit (BD PharMingen). Briefly, 1×105 were extensively washed with PBS and incubated
treated cells were incubated with Annexin Ⅴ/ with horseradish peroxidase conjugated secondary
propidium iodide for 15 min at room temperature. anti-mouse antibody or anti-rabbit antibody
The cells were then analyzed by flow cytometry (dilution 1:2,000, BioRad). After additional
using two-color fluorescence-activated cell sorting washes with PBS, antigen-antibody complexes
analysis (BD LSR Ⅱ). were visualized with the enhanced
Luciferase reporter assay- The pMIR-reporter chemiluminescence kit (Pierce).
luciferase vector containing the 3’-UTR of Bak1 Cell Viability Assay- A total of 5 x 103~1 x 104
and the empty vector were generous gifts from Dr. cells/well were seeded in 96-well plates.
deVere White (20). For the luciferase assay, cells Twenty-four hrs later, the medium was replaced
at the density of 1.2 x 105 per well in 24-well with fresh medium with or without Taxol and
4
incubated for 48 hrs, respectively. Cell viability SKBr3-TRP, which were derived from
was determined using CellTiter 96 Aqueous One MD-MB-436 and SKBr3 breast cancer cell lines
Solution Cell Proliferation Assay kit (Promega) as respectively. More than two-fold higher expression
previously reported (21). of miR-125b was found in Taxol-resistant 436TRP
Statistical analysis- Statistical evaluation for and SKBr3-TRP cells compared to their parental
data analysis was determined by unpaired MDA-436 and SKBr3 cells respectively (Fig. 1C),
Student’s t-test. All data were shown as the mean indicating that the correlation between miR-125b
± standard error (SE). A statistical difference of P and Taxol resistance is not a single cell line
< 0.05 was considered significant. specific effect. These results suggested that the
differential expression profiles screened by the
RESULTS miRNA array are reliable, and some of them may
play a role in Taxol resistance.
Profiles of miRNAs in Taxol-sensitive and Involvement of miR-125b in Taxol resistance
resistant cancer cells. To search for the critical in breast cancer cells. We investigated the role
D
miRNAs involved in Taxol resistance, the total of miR-221, miR-222 and miR-125b in Taxol- o
w
n
RNA extracted from Taxol-resistant 435TR1 and induced cytotoxicity. Compared to negative lo
a
d
e
435TRP cells and their Taxol-sensitive parental control, overexpression of miR-221 and miR-125b d
fro
MDA-435 cells were hybridized with miRNA markedly inhibited Taxol-induced cell cytotoxicity m
h
array chips. Eight miRNAs were up-regulated in in MDA-435 cells (Fig. 2A). Since miR-125b ttp
://w
Taxol-resistant cells by more than 1.5 folds, and showed a stronger inhibition to Taxol-induced w
w
thirteen miRNAs were down-regulated in cytotoxicity, we first focused on miR-125b and .jb
c
.o
Taxol-resistant cells by more than 1.5 folds (Fig. further investigated its role and mechanism in rg
b/
1A and Table S1). Notably, the profiling results are Taxol resistance. y
g
u
correlative with the reported functions of miRNAs. To confirm and extend our findings, two es
t o
n
For example, miRNAs with oncogenic function, more breast cancer cell lines, MDA-231 and O
c
such as miR-100 (22), miR-125b (20, 23-26), BT474, were used to investigate the effect of tob
e
miR-106b (27), miR-221 and miR-222 (28, 29), overexpression of miR-125b on Taxol resistance. r 5
, 2
0
were up-regulated, while miRNAs with tumor Overexpression of miR-125b markedly inhibited 1
7
suppressor functions, such as miR-16 (30, 31), Taxol-induced cytotoxicity in all of the cell lines
miR-27a (32), miR-20a (33), miR-29a/b (34), and tested (Fig. 2B). Moreover, overexpression of
miR-146a/b (35), were down-regulated in miR-125b dramatically decreased Taxol-induced
Taxol-resistant cells. To further validate the cytotoxicity and increased Taxol resistance in a
miRNA array results, we randomly selected four miR-125b dose-dependent manner in MDA-435
miRNAs, miR-923, miR-125b, miR-221 and cells (Fig. 2C, top). Furthermore, the expression of
miR-222 from the up-regulated miRNAs and miR-125b (Fig. 2C, bottom inset) decreased
measured their expression levels by real-time PCR. Taxol-induced cytotoxicity and increased Taxol
The results of real-time PCR for all of the four resistance under various concentrations of Taxol
selected miRNAs were consistent with the miRNA treatment in MDA435 cells (Fig. 2C, bottom).
array results (Fig. 1B). Furthermore, we examined Similar experiments were performed in MDA-231
the expression of miR-125b in two more cells with similar results obtained (Fig. 2D).
Taxol-resistant cancer cell variants, 436TRP and While overexpression of miR-125b confers
5
Taxol resistance to cells, we asked whether cells were detected in pre-miR-125b transfected
downregulation of miR-125b may increase Taxol MDA-435 and MDA-231 cells compared to
sensitivity. miR-125b antisense (anti-miR-125b) negative controls (Fig. 3D). Furthermore,
was expressed in Taxol-resistant 435TR1 cells, knock-down of endogenous miR-125b enhanced
followed by treatment with increasing Taxol-induced apoptosis in Taxol-resistant
concentrations of Taxol for 48 hrs. We found that 435TRP cells (Fig. S2). These results indicated
the knockdown of miR-125b by anti-miR-125b that miR-125b confers Taxol resistance in cancer
increased Taxol-induced cytotoxicity in these cells cells by blocking Taxol-induced apoptosis.
known to be highly resistant to Taxol (Fig. S1). Bak1 is a direct target of miR-125b in breast
These results clearly demonstrate that miR-125b cancer cells. Since miR-125b is capable of
plays an important role in Taxol resistance in suppressing Taxol-induced apoptosis and
breast cancer cells. increasing the resistance of cancer cells to Taxol,
Involvement of miR-125b in Taxol-induced we searched miRNA databases for potential
apoptosis. Taxol is known to exert its cancer cell miR-125b targets that may possibly contribute to
D
killing effect through induction of apoptosis (36). Taxol resistance. The three public miRNA o
w
n
Next, we examined whether overexpression of databases (targetscan.org, pictar.bio.nyu.edu, and lo
a
d
e
miR-125b was capable of inhibiting Taxol-induced microrna.org) all predicted that Bak1 might be a d
fro
apoptosis. MDA-435 cells were transfected with target for miR-125b, and the 3’-UTR of Bak1 m
h
pre-miR-negative or pre-miR-125b, and then contains a highly conserved binding site from ttp
://w
treated with increasing concentrations of Taxol. position 629 to 635 for miR-125b (Fig. S3). To w
w
Cell morphological change was observed with determine whether miR-125b could target Bak1 in .jb
c
.o
microscope, showing markedly less cellular debris breast cancer cells, we transfected the rg
b/
and non-viable cells in miR-125b transfected cells pre-miR-125b into MDA-435, MDA-231, MCF7 y
g
u
compared with negative controls (Fig. 3A). Poly and SKBr3 cells respectively. The expression of es
t o
n
(ADP-ribose) polymerase (PARP) is an important miR-125b significantly down-regulated Bak1 O
c
marker of apoptosis and it is cleaved downstream protein in a dose-dependent manner in all tested tob
e
of caspases when cellular apoptosis happens (37, cells (Fig. 4A and 4B). Moreover, knock-down of r 5
, 2
0
38). We next detected the protein levels of the miR-125b up-regulated the expression of Bak1 in 1
7
cleaved PARP and total PARP by western bloting both MDA-231 and 435TRP cells with high
in pre-miR-125b transfected MDA-435 and expression of endogenous miR-125b (Fig. 4C).
control cells after Taxol treatment. Compared to These results demonstrate that Bak1 is a target of
the negative control, the protein level of cleaved miR-125b in breast cancer cells.
PARP was dramatically reduced in pre-miR-125b We therefore investigated whether miR-125b
transfected cells (Fig. 3B). The activity of caspase directly targets the 3’-UTR of Bak1 mRNA, we
3 was further detected using Caspase-Glo® 3/7 performed luciferase reporter analysis by
Assay system in pre-miR-125b transfected co-transfecting a vector containing pMIR
MDA-435 cells after Taxol treatment for 24 hrs. Reporter-luciferase fused with or without the 3′
Lower caspase 3 activity was found in miR-125b UTR of Bak1 mRNA, and pre-miR-125b or
transfected cells compared to the control cells (Fig. pre-miR-Negative. Overexpression of miR-125b
3C). Moreover, apoptosis assays with Annexin V decreased the luciferase activity of the reporter
staining revealed that significantly less apoptotic with 3′ UTR of Bak1 by about 60% in both
6
MDA-435 and MDA-231 cells (Fig. 4D, left). Bak1 plays a critical role in Taxol-induced
However, no inhibitory effect of miR-125b on the apoptosis. Bak1, together with Bax, is an
activity of the reporter without 3′ UTR of Bak1 ultimate death effecter of apoptosis triggering cues
was detected (Fig. 4D, right), indicating that Bak1 (39). Although Bak1 has been proven to play a
mRNA is a direct target of miR-125b. critical role in apoptosis, its role in Taxol-induced
To examine whether miR-125b has the cell cytotoxicity and Taxol sensitivity has not been
capacity to suppress the expression of Bak1 at studied. To investigate the association of Bak1 and
mRNA level. Quantitative PCR was performed to Taxol-induced cell cytotoxicity, we transfected
detect Bak1 mRNA expression in MDA-435 and Bak1-specific siRNA into MDA-435 cells, and
MDA-231 cells after transfection with then treated these cells with increasing
pre-miR-125b or pre-miR-Negative. We found that concentrations of Taxol. Compared to negative
miR-125b decreased the expression of Bak1 control, Bak1 siRNA efficiently knocked down
mRNA with a similar inhibitory efficiency to the Bak1 expression (Fig. 5A, inset), and decreased
Bak1-specific siRNA in both cell lines, indicating the inhibition of cell viability by Taxol (Fig. 5A).
D
that miR-125b inhibits the expression level of Similar experiments were performed in MDA231 o
w
n
Bak1 mRNA (Fig. 4E). To explore the possibility cells and the results were similar (Fig. S4). lo
a
d
e
that miR-125b decreases the stability of Bak1 Moreover, compared to control cells, si-Bak1 d
fro
mRNA, MDA-435 cells were transfected with or transfected MDA-435 cells showed much less m
h
without pre-miR-125b and then treated with cellular debris and non-viable cells (Fig. 5B). ttp
://w
transcription inhibitor Actinomycin D. The PARP cleavage was also dramatically reduced in w
w
endogenous Bak1 mRNA level was determined by si-Bak1 transfected MDA-435 and MDA-231 cells .jb
c
.o
qRT-PCR. We found that after treatment of after treatment with 20 or 40 nM Taxol for 48 hrs rg
b/
Actinomycin D for 10 hrs, the Bak1 mRNA (Fig. 5C). Cellular apoptosis were further detected y
g
u
expression level was lower in miR-125b in MDA-435 and MDA-231 cells by Annexin Ⅴ es
t o
n
overexpressing cells compared to that of the staining, the results revealed markedly less O
c
control cells (Fig. 4F), indicating that miR-125b apoptotic cells in si-Bak1 transfected cells (Fig. tob
e
decreases the stability of Bak1 mRNA. To test 5D). These results indicate that the knockdown of r 5
, 2
0
whether an increased expression of miR-125b Bak1 could efficiently suppress Taxol-induced cell 1
7
would correlate with a decreased expression of cytotoxicity via the inhibition of Bak1-mediated
Bak1 protein and mRNA levels in Taxol-resistant apoptosis.
cancer cells, we performed Western blotting and Restoring the expression of Bak1 recovers
quantitative real-time PCR to detect the expression Taxol-sensitivity in counteracting miR-125-
of Bak1 in 435TR1, 435TRP and their parental mediated Taxol resistance. Since Bak1, a direct
MDA-435 cells. Compared with the parental cells, target of miR-125b, plays a critical role in
decreased expression of Bak1 was found in Taxol-induced apoptosis, we next investigated
Taxol-resistant 435TR1 and 435TRP cells at both whether miR-125b affects Taxol resistance through
protein and mRNA levels (Fig. 4G). These results suppression of Bak1 expression. We therefore
further support that Bak1 is a target of miR-125b, transfected pre-miR-125b, or pre-miR-125b in
and the loss of Bak1 expression in Taxol-resistant combination with anti-miR-125b into MDA-435
cells may mediate the role of miR-125b in Taxol and MDA-231 cells, and then we treated the cells
resistance. with indicated concentrations of Taxol for 48 hrs.
7
Transfection with the pre-miR-125b alone in chemoresistance, such as to Taxol. In this study,
decreased the expression of Bak1 (Fig. 6A, top) we compared the differential profiles of miRNAs
and increased the resistance of these cells to Taxol between Taxol-resistant and their parental
and suppressed apoptosis in both of cell lines (Fig. Taxol-sensitive MDA-435 cells. We found that
6A, bottom). Interestingly, transfection with the most of the miRNAs that are highly expressed in
miR-125b antisense, anti-miR-125b, restored the Taxol-resistant cells are previously reported to
expression of Bak1 (Fig. 6A, top), and recovered function as oncogenes, such as miR-221 (27, 28),
Taxol-induced cytotoxicity (Fig. 6A, bottom) and miR-222 (28, 29), miR-100 (22), and miR-106b
apoptosis (Fig. 6B, Fig. S5). Moreover, with (27). Of the lower expressed miRNAs, most of
recovery of Bak1 expression by miR-125b them are previously reported as tumor suppressors,
antisense, PARP cleavage was also restored in such as miR-16 (30, 31), miR-20a (33), miR-29a/b
pre-miR-125b expressing cells when treated with (34), and miR-146a/b (35). Taxol resistance is a
Taxol (Fig. 6C). phenotype of malignant cancer cells; therefore, the
To further confirm these results, we findings of our microRNA assay are in line with
D
co-transfected pre-miR-125b plus the control the reported functions of miRNAs, suggesting our o
w
n
vector or a Bak1 expression vector into MDA-435 profiling results are reliable. lo
a
d
e
cells, and then treated these cells with Taxol. We verified the differential expression levels d
fro
Transfection of pre-miR-125b alone decreased the of miR-221, miR-222 and miR-125b, and m
h
expression of Bak1 and increased the resistance of examined the effects on Taxol-induced cytotoxicity. ttp
://w
these cells to Taxol (Fig. 6D), however, restoring We found that miR-125b and miR-221 markedly w
w
expression of Bak1 by co-transfecting inhibited Taxol-induced cytotoxicity in breast .jb
c
.o
pIRES2-EGFP/Bak1 recovered the Taxol cancer cell lines. The inhibitory effect was found rg
b/
sensitivity (Fig. 6D). Of note, the overexpression to be dose-dependent, over a wide range of Taxol y
g
u
of miR-125b had no effect upon the expression of concentrations. Moreover, antisense to miR-125b es
t o
n
exogenously expressed Bak1. This can be increased Taxol-sensitivity in Taxol-resistant cells, O
c
explained by the lack of 3’-UTR in the clearly demonstrating that miR-125b plays a tob
e
exogenously expressed Bak1 mRNA. Taken critical role in the development of Taxol resistance. r 5
, 2
0
together, these results clearly demonstrate that It was reported that miR-125b could be 1
7
Bak1 plays an important role in breast cancer cells transactivated by NF-κB p65 subunit through
Taxol sensitivity and overexpression of miR-125b binding to the promoter of miR-125 (40, 41). We
confers Taxol resistance at least partially through also found that compared to that of the parental
the direct suppression of Bak1 expression. MDA-435 cells, Taxol-resistant cells showed
increased trascriptional activity of NF-κB (Fig. S6),
implying that activation of NF-κB by long term
DISCUSSION Taxol treatment might be a mechanism for
Taxol-mediated upregulation of miR-125b. Further
Many miRNAs have been identified as study is needed to validate this potential
having an oncogenic or tumor suppressor-like mechanism.
function，shown to be involved in cell proliferation, We also indentified a lower percentage of
differentiation, apoptosis and drug resistance apoptotic cells in miR-125b transfected cells by
(14-18). However, little is known about their role Annexin V staining after treatment with Taxol,
8
further confirmed by immunoblotting of the been identified. One recent study showed that
apoptotic marker, cleaved PARP, and through tumor suppressor p53 is a direct target of
detection of caspase 3 activity. These results miR-125b, and suppression of p53 gene expression
suggest that the anti-apoptotic effect is a key through overexpression of miR-125b inhibited
mechanism of miR-125b-mediated Taxol p53-dependent apoptosis in human neuroblastoma
resistance in breast cancer cells. cells and human lung fibroblast cells (43). Another
Apoptosis is a predominant mechanism by report showed that ErbB2 and ErbB3 were both
which cancer chemotherapeutic agents such as targets of miR-125b in breast cancer SKBr3 cells
Taxol to kill cancer cells (42). Bak1 is an ultimate (44). Conversely, Shi et al. showed that there was
death effecter of apoptosis triggering cues. It is no miR-125b-mediated down-regulation of ErbB2
localized to the cytoplasmic side of the and ErbB3 in miR-125b-transfected LNCaP and
mitochondrial membrane, tethered by a cds1 prostate cancer cells (20). Consistent with the
membrane-bound C-terminal helix, and is held latter report, we also did not find the
inactive by forming heterodimeric complexes with down-regulation of ErbB2 by miR-125b in breast
D
the anti-apoptotic Bcl2 family member Mcl1 (39). cancer BT-474, BT-474M1 and SKBr3 cells (Fig. o
w
n
Bak1 was recently found to be a target of S7). In line with these findings, miR-125b was lo
a
d
e
miR-125b and implicated in androgen-independent reported to have complicated functions as either d
fro
growth in prostate cancer cells (20). Here, we oncogene-like or tumor suppressor-like in different m
h
demonstrate that Bak1 is a direct target of cancer types or cell lines. miR-125b is not only ttp
://w
miR-125b in breast cancer cells. Moreover, we elevated in pancreatic cancer, oligodendroglial w
w
show that Bak1 enhanced Taxol-induced tumors, prostate cancer, myelodysplastic .jb
c
.o
cytotoxicity and apoptosis. To our knowledge, this syndromes and acute myeloid leukemia, but also rg
b/
is the first report to demonstrate a direct link promotes cell proliferation in prostate cancer cells, y
g
u
between Bak1 expression and cancer cell Taxol enhances invasive potential in urothelial es
t o
n
sensitivity. Importantly, we found that restoring carcinomas and suppresses p53-dependent O
c
the expression of Bak1, either by anti-miR-125b or apoptosis in human neuroblastoma cells (20, 23-26, tob
e
exogenously Bak1 expression, recovered Taxol 45). However, miR-125b has been reported to be r 5
, 2
0
sensitivity in counteracting miR-125-mediated down-regulated in ovarian carcinoma, thyroid 1
7
down-regulation of Bak1 and Taxol resistance. carcinoma and oral squamous cell carcinoma, and
Thus, miR-125b expression contributes to Taxol has been shown to inhibit cell proliferation and
resistance through suppression of Bak1 expression. cell cycle progression in these cancers (46-48).
These results indicate that miR-125b-Bak1 axis is Similar phenomenon of duality of function for
critical for Taxol resistance in breast cancer cells miRNA in different types of cancers has also been
and may potentially serve as a therapeutic target to found on miR-29a (49) and miR-17-92 cluster (50).
overcome Taxol resistance. This study represents One possible explanation is the known diverse
one of the first attempts to explore the role of nature of miRNA target genes. The net effect of
microRNA in cancer cell Taxol resistance. This changes in the expression of a miRNA appears to
novel finding provides a unique insight into the be the sum of all impacts on its targets in a
molecular mechanism of miR-125b-mediated cell-type-specific and phenotype-specific manner
Taxol resistance. (49). Thus overexpression or knockdown of a
To date, several targets of miR-125b have miRNA might have diverse effects which depend
9
on the types of cancer cells. and not of melanoma origin [56]. Nevertheless, we
Recently, miR-125b was reported to exhibit confirmed all of the results from MDA-MB-435
under-expressed in breast cancer tissues (51), cells with another breast cancer cell line
however, the exact association of miR-125b MDA-MB-231 and we further confirmed the key
expression and breast tumor progression is still findings with breast cancer cell lines
unclear. Two recent studies reported that MDA-MB-436, SKBr3, BT474, and MCF-7.
miR-125b may act as an oncogene in human breast Taken together, we have shown for the first
cancer cells. One study reported that miR-125b time that miR-125b is up-regulated in
promoted cell proliferation and decreased the Taxol-resistant cells, with the overexpression of
antitumor effects of 1,25(OH)(2)D(3) in breast miR-125b causing a marked inhibition of
cancer MCF7 cells (52); Another study showed Taxol-induced apoptosis, and increased resistance
that compared to nontumorigenic cancer cells, to Taxol. We demonstrate that Bak1 is a direct
miR-125b is overexpressed in highly tumorigenic target of miR-125b in breast cancer cells.
human breast cancer stem cells (53). In our study, Moreover, we first showed that Bak1 plays an
D
we show that miR-125b inhibited Bak1 and important role in Taxol sensitivity of breast cancer o
w
n
increased Taxol resistance, acts primarily as an cells. Thus, miR-125b confers Taxol resistance lo
a
d
e
oncogene in breast cancer MDA-435, MDA-231, through suppression of Bak1 expression and may d
fro
SKBr3, and BT474 cells. However, further potentially serve as a therapeutic target for m
h
investigations with large patient samples and well overcoming Taxol resistance in human breast ttp
://w
designed laboratory studies should be performed cancer. These novel findings have important w
w
in the future to clarify the role of miR-125b in implications in the development of targeted .jb
c
.o
human breast cancer development. therapeutics for overcoming Taxol resistance in rg
b/
The origin of MDA-MB-435 cells has human breast cancer. y
g
u
recently been called into question [54-55]. es
t o
n
However, a latest literature indicated that O
c
MDA-MB-435 cells are in fact breast cancer cells tob
e
r 5
, 2
0
1
7
REFERENCES

1. Henley, D., Isbill, M., Fernando, R., Foster, J.S., and Wimalasena, J. (2007) Cancer Chemother
Pharmacol 59, 235-249
2. Tan, M., and Yu, D. (2007) Adv Exp Med Biol 608, 119-129
3. Chen, L.P., Cai, S.M., Fan, J.X., and Li, Z.T. (2002) Gynecol Oncol 56, 231-234
4. Gottesman, M.M., and Pastan, I. (1993) Annu Rev Biochem 62, 385-427
5. Panda, D., Miller, H.P., Banerjee, A., Ludueña, R.F., and Wilson, L. (1994) Proc Natl Acad Sci U S A
91, 11358-11362
6. Martello, L.A., Verdier-Pinard, P., Shen, H.J., He, L., Torres, K., Orr, G..A., and Horwitz, S.B. (2003)
Cancer Res 63, 1207-1213
7. Goncalves, A., Braguer, D., Kamath, K., Martello, L., Briand, C., Horwitz, S., Wilson, L., and Jordan,
M.A. (2001) Proc Natl Acad Sci 98, 11737-11742
10