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Synuclein gamma compromise spindle assembly checkpoint and renders resistance to antimicrotubule
drug
By
Suyu Miao1, Kejin Wu2, Bo Zhang1, Ziyi Weng2, Mingjie Zhu3, Yunshu Lu2, Ramadas Krishna4, and
Yuenian Eric Shi1,5
1Department of Surgical Oncology, The First Affiliated Hospital of Nanjing Medical University, Nanjing,
China
Departments of 2General Surgery and 3Pathology, XinHua Hospital
Shanghai Jiaotong University School of Medicine 4Centre for Bioinformatics, School of Life Science, Pondicherry University.
5The Feinstein Institute for Medical Research, New York, USA
Y.E.Shi was supported in part by grants 81230054 and 81071819 from State Key Program of National
Natural Science Foundation of China; a grant from National Key Technology R&D Program for the 12th
Five-year Plan of china (2013BAI01B06); and a grant from State Key Laboratory of Reproductive Medicine.
K.Wu was supported in part by a grant 81102016 from National Natural Science Foundation of China
The first two authors contribute equally
Corresponding authors: Dr. Yuenian Eric Shi and Kejin Wu
E-mail: [email protected] (Yuenian Eric Shi)
[email protected] (Kejin Wu)
The authors declare no conflicts
Running Title: Synuclein gamma and antimicrotubule drug resistance
Key words: Synuclein gamma, antimicrotubule drug, spindle assembly checkpoint, BubR1, drug resistance
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Abbreviations: APC, anaphase-promoting complex. MCC, mitotic checkpoint complex. MT,
microtubule. pCR, pathologic complete response. SAC, spindle assembly checkpoint. SNCG,
synuclein gamma
Abstract
Defects in the spindle assembly checkpoint (SAC) have been proposed to contribute to the chromosomal
instability in human cancers. One of the major mechanisms underlying antimicrotubule drug (AMD)
resistance involves acquired inactivation of SAC. Synuclein gamma (SNCG), previously identified as a
breast cancer specific gene, is highly expressed in malignant cancer cells but not in normal epithelium. Here,
we show that SNCG is sufficient to induce resistance to AMD-caused apoptosis in breast cancer cells and
cancer xenografts. SNCG binds to spindle checkpoint kinase BubR1 and inhibits its kinase activity.
Specifically, the C-terminal (Gln106-Asp127) of SNCG binds to the N-terminal TPR motif of BubR1.
SNCG-BubR1 interaction induces a structure change of BubR1, attenuates its interaction with other key
checkpoint protein of Cdc20, and thus compromise SAC function. SNCG expression in breast cancers from
patients with a neoadjuvant clinical trial showed that SNCG-positive tumors are resistant to chemotherapy-
induced apoptosis. These data show that SNCG renders AMD resistance by inhibiting BubR1 activity and
attenuating SAC function.
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Introduction
The microtubule (MT) cytoskeleton is an effective and validated target for cancer chemotherapeutic
drugs. Currently, AMD taxane, e.g. docetaxel, is the first-line chemotherapeutic agent to treat patients with
locally advanced or metastatic breast cancer and is also the effective chemotherapeutic agent in clinical use
in six different cancers (breast, ovarian, non-small cell lung, prostate, gastric, and head and neck)[1] .
Cytotoxicity stems from its ability to promote tubulin polymerization and formation of stable microtubules.
The stabilized microtubules are resistant to disassembly by physiologic stimuli, so cells accumulate
disorganized arrays of microtubules. This results in arresting the cell in the G2-M phase, which ultimately
results in apoptosis. Nearly half of the treated breast and ovarian cancer patients, however, do not respond to
this microtubule disrupting chemotherapy[2] . Identification of cellular factors that are associated with the
sensitivity to AMD treatment would have great clinical implications.
Although general mechanisms of drug resistance may apply to AMD resistance[3][4], more specifically,
the current research paradigm on AMD resistance focus on acquired β-tubulin mutations and altered
expression of β-tubulin isotypes[5][6]. Since tubulin is the structural protein of MT and is supposed to be the
main target of docetaxel action, alternations of tubulin expression are thought to induce docetaxel resistance.
However, it is not clear what effect the expression of a particular β-tubulin isotype, or acquisition of point
mutations has on the stability of MT and their relationship to docetaxel resistance. Furthermore, several
recent studies searching for biomarkers predictive of clinical response to taxane are largely contradictory
and, in any case, very limited on the predictive role that tubulin characteristics could have biological effects
of these drugs[7][8]. One of the mechanisms underlying AMD resistance involves acquired inactivation of
mitotic checkpoint function. AMD works by perturbing spindle assembly, which activates SAC, resulting in
cells arrested in the mitotic phase without entering anaphase[9] . Prolonged treatments with these agents lead
to cell death by undergoing apoptosis. Since AMD is thought to induce mitotic catastrophe, which activates
SAC, if SAC is defective or inhibited in cancer cells, the cells will arrest transiently and proceed through
mitosis without undergoing apoptosis. In fact, cancer cells can resist such killing by premature exit, before
cells initiate apoptosis, due to a weak or inactivated SAC[10] . Although genetic mutations in the key SAC
components, e.g. BubR1, are associated with the cancer susceptible disorder mosaic variegated
aneuploidy[11], mutations in the known SAC genes are not very often seen in human carcinomas, suggesting
that SAC inactivation in human tumors may also be driven by an epigenetic mechanism.
We previously identified a breast cancer specific gene BCSG1, also known as synuclein γ (SNCG)[12].
Synucleins are a family of small proteins consisting of 3 known members, synuclein α (SNCA), synuclein β
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(SNCB), and SNCG[13] . While synucleins are highly expressed in neuronal cells and have been specifically
implicated in neurodegenerative diseases[14][15], SNCG is not clearly involved in neurodegenerative
diseases but is primarily involved in neoplastic diseases. So far, the abnormal expression of SNCG protein
has been demonstrated in many different malignant diseases, including breast[12][16][17], liver[18][19],
esophagus[18] , colon[18][20][21], gastric[18] , lung[18], prostate[18], pancreas[22], bladder[23], cervical
cancers[18] , ovarian cancer[24], and glial tumors[25]. In these studies, SNCG protein is abnormally
expressed in a high percentage of tumor tissues but rarely expressed in tumor-matched nonneoplastic
adjacent tissues. The clinical relevance of SNCG expression on breast cancer prognosis was confirmed in
clinical follow-up studies[16][17]. Patients with an SNCG-positive tumor had a significantly shorter disease-
free survival and overall survival compared with the patients with no SNCG expression. SNCG is a new
unfavorable prognostic marker for breast cancer progression and a potential target for breast cancer
treatment. At the cellular level, SNCG increases metastasis[26] and hormone-dependent tumor growth[27-
29], and promotes genetic instability[30][31]. Investigations aimed to elucidate the molecular mechanisms
underlying the oncogenic functions of this protein revealed that SNCG functions like a tumor specific
chaperone and regulates many pathways in cancer progression, which include cell motility26 and estrogen
receptor signaling[27-2829][32].
Previous studies conducted through yeast two-hybrid screening revealed an interaction of SNCG with
the mitotic checkpoint kinase BubR1[30]. BubR1 was originally characterized as a kinase that controls the
activation of the anaphase-promoting complex (APC) by binding and inhibiting Cdc20[33], the major APC
regulatory protein. BubR1 is recognized as an essential component of the mammalian checkpoint machinery
that monitors the proper assembly of the mitotic spindle. Since the working mechanism of AMD heavily
relies on the normal function of the SAC in which BubR1 is a critical component, we reason that the SNCG-
BubR1 interaction may represent a novel mechanism for inactivation of the mitotic checkpoint and thus
renders AMD resistance. In this study, we investigated the role of SNCG on BubR1 function, its interaction
with other key members in SAC, and on inhibition of docetaxel-induced mitotic checkpoint function. The in
silico interaction between SNCG with the crystal structure of human BubR1 was analyzed. We also
investigate whether SNCG is a new biomarker for predicting resistance of breast cancer patients to docetaxel
in neoadjuvant treatment.
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METHODS
Materials and Cells. Docetaxel was acquired from Northcarolina Chemlabs. Nocodazole was acquired from
Sigma. A stock solution of docetaxel (dissolved in 5% Tween 80, 5% DMSO in PBS) was stored at -80°C
before use. For in vitro use, docetaxel was diluted in serum-free medium at the required concentration. For in
vivo use, docetaxel was diluted in normal saline to a final dose of 25 mg/kg in 150 μl per injection. All of the
cell lines used in this study were originally obtained from the American Type Culture Collection as we
described before[27,28]. No authentication was done. SNCG stably transfected MCF-7 (MCF-S6, MCF-
S2)[27,28] and MDA-MB-435 (SNCG-435-3)[26] cells were established in 2003 and 1999. SNCG
knockdown MDA-MB-231 and SKBR3 cells were previously established in 2010[32]. Docetaxel resistant
MCF-7 and MDA-MB-231 cells were generated by prolonged and repeated exposure to increasing doses of
docetaxel. Cells were initially exposed to 50 nM for 1 week, increasing to 200 nm for another 1 week. After
this point, the cells were exposed to 0.6 μM docetaxel for 3 months.
Determination of apoptotic cells and mitotic index. Apoptotic cells were determined by propidium iodide
staining and flow cytometry as we previously described[32]. For mitotic index, cells grown on coverslips
were treated with docetaxel (80 nM, 20 h), fixed with 70% ethanol, and incubated with 5 mg/ml of anti-
MPM-2 antibody (in PBS/0.5% BSA for 1 h) that recognizes mitosis-specific phosphorylated proteins
(Upstate). Cells were then washed and incubated with Alexa Fluor 488 conjugated antibody (Invitrogen)
diluted in PBS containing 50 mg/ml RNaseA and 50 mg/ml propidium iodide and analyzed on a LSRII
(Becton Dickinson).
In vitro BubR1 kinase activity. Cellular extracts from docetaxel-arrested (80 nM, 30 h) cells were
immunoprecipitated (IP) with specific anti-BubR1 antibody (Santa Cruz, sc-54). The IP complex was
washed twice with IP buffer (1% Triton X-100, 10 mM Tris pH 7.4, 0.5% NP-40, 150 mM NaCl, 1 mM
EDTA, 1 mM EGTA) containing protease inhibitor cocktail. BubR1 immunoprecipitates were incubated at
room temperature for 30 min with 25 mM Hepes (pH 7.5), 10 mM MgCl2, 200 μM ATP, and 1
μCi[γ32P]ATP, and 100 μg histone H1 as an exogenous substrates. The kinase reaction was terminated by
addition of SDS sample buffer and boiling. The phosphorylated H1 was separated by SDS-PAGE and
detected by exposing to a screen of PhosphoImager.
Molecular docking study. The in silico interaction study between SNCG and the crystal structure for N-
terminal region of human BubR1 was carried out using the easy interface option of HADDOCK online
server. The GIG motif of BuBR1 (146-148) and Glu110, Glu117 and Asp127 of SNCG were defined as the
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initial active site residues and residues surrounding them within the radius 6.5 Å were defined as the passive
residues. In order to confirm the interactions between the C-terminal region residues of SNCG and N-
BubR1, the docking study between the SNCG C-terminal region and BubR1 was also carried out using
HADDOCK employing the same parameters as provided above. The PyMol molecular visualization tool
version 0.99 was used to analyze and to prepare the images and DimPlot was used to plot the interactions
between SNCG and BubR1.
Molecular Dynamics study. Molecular Dynamics simulations of SNCG-BubR1 and C-terminal region of
SNCG-BubR1 structure complexes and the stability of the interactions were analyzed using the molecular
dynamic simulation studies carried out using the GROMACS 4.0 software package. Initially, the protein
structures were typed with the OPLS all atom force field and later they were placed in a cubic box and
solvated with the water molecules using the spc216 water models. Later the energy of the structures were
minimized using the steepest descendent method and simulation was carried out for 3 nanoseconds at a room
temperature, by employing the LINCS constraint and Particle Mesh electrostatics method along with the
Berenson temperature and pressure coupling system (maintaining the temperature and pressure of the
system). The whole procedures were carried out in a HP workstation (HP xw4600) provided with the Intel
core 2 due core processors. The programs built within the GROMACS package was used for computing
RMSD and other calculations. The RMSD and potential energy maps were created using the XMGRACE
software.
Tumor growth in athymic nude mice. A nude mouse tumorigenic assay was performed as we previously
described[28][32]. Briefly, approximately 3 x106 (MCF-7) or 1 x 106 (MDA-MB-435) cells were injected
into a 6-week old female athymic nude mouse. Each animal received two injections, one on each side, in the
mammary fat pads between the first and second nipples. For MCF-7 tumor xenografts, 17β-estradiol pellets
(0.72 mg/pellet, 60-day releasing, Innovative Research of America, Toledo, OH) were implanted
subcutaneously one day before the injection of the cells. When tumor xenografts were established, mice
bearing tumors were randomly allocated to different treatment groups. Each group has 8 mice. For drug
administration, docetaxel was administrated at 25 mg/kg, i.p., and 1 time/5-days for 25 days. Tumor size was
determined at weekly intervals and only measurable tumors were used to calculate the mean tumor volume
for each tumor cell clone at each time point.
Patients. Thirty women with core biopsy-proven infiltrating breast cancer (staged as T2-4N0-2M0 based on
UICC TNM classification) were enrolled on study at Xinhua Hospital, Shanghai Jiaotong University School
of Medicine. The median age of 30 breast cancer patients was 52 (32–70 years old). After signing informed
consent, patients received standard 3 cycles TE chemotherapy (Docetaxel 75mg/m2 IV d1 and epirubicin
60mg/m2 IV d1, every three weeks). Patients were required to have adequate metabolic functions before
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every cycle chemotherapy and fertile women had to use effective contraception. All patients accepted the
modified radical mastectomy 2 weeks after the last cycle of chemotherapy.
Immunohistochemical analysis. As previously described[17], deparaffinized human breast sections (5μm
thick) were treated with H2O2, trypsin, and blocked with normal goat serum. Sections were incubated with
specific mouse monoclonal antibodies against with ER, PR, p53, HER-2 and Ki67 (Antibody Diagnostica
Inc.) followed by the incubation with the biotin-conjugated secondary antimouse antibodies (DAKO). The
colorimetric detection was performed by using a standard indirect streptavidin-biotin immunoreaction
method by DAKO’s Universal LSAB Kit. SNCG was detected by using affinity purified sheep anti-SNCG
polyclonal antibody (Chemicon International Inc.). Approximately 100 tumor cells per field were counted
under a Nikon microscope at 200×amplification and eight fields were randomly selected in each slide. The
negative cases were confirmed with two independent experiments. All the slides were scored by a breast
cancer pathologist without knowledge of the clinical outcome.
TUNEL staining (ApopTag peroxidase in situ Apoptosis Detection Kit from Chemicon International)
for apoptotic index was scored by point counting of at least 500 cancer cells, and results were presented as
percent-positive tumor cells.
Statistical analyses. For in vitro and tumor xenograft studies, results were reported as the mean ± SD for
typical experiments done in three replicate samples and compared by the Student’s t test. All experiments
were done at least twice to ensure reproducibility of the results. Clinical data were analyzed by using SPSS
17.0. Results were reported as the mean ± SD. Differences in tumor response between the treatment groups
were compared using the χ2 test. All statistical analyses were two-sided, and comparisons made in which p <
0.05 were considered statistically significant.
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RESULTS
SNCG confers cellular resistance to docetaxel-induced apoptosis. We determined whether alternation
of SNCG expression in breast cancer cells affects cell response to docetaxel-mediated cytotoxicity. We first
used SNCG negative MCF-7 and its stably SNCG transfected clones[27][28]. Stable expression of SNCG
compromised the sensitivity to docetaxel-induced killing in MCF-7 cells in concentration-dependent manner
(0.05-0.8 μM; data not shown). As demonstrated in Fig. 1A, while docetaxel treatment induced a 45% and
52% of apoptosis in parental MCF-7 cells and neo transfected control MCF-neo1 clone, respectively,
expression of SNCG rendered cellular resistance to docetaxel, which resulted in only 22% and 18% of
apoptotic cells in SNCG stably transfected MCF-S2 and MCF-S6 clones, respectively. Since SNCG activates
ERα transcriptional activity[27][28] and taxol downregulated ERα expression in MCF-7 cells, it is possible
that the observed SNCG-mediated anti-docetaxel effect in MCF-7 cells is mediated indirectly by stimulation
of ERα activity. To exclude this possibility, we used previously established ER-negative and SNCG stably
transfected MDA-MB-435 cells[26]. The similar anti-apoptotic effect of SNCG was also observed in
docetaxel-treated SNCG transfected MDA-MB-435 cells. Compared with parental MDA-MB-435 and its
neo control neo-435-1 cell, the stable SNCG transfected clone SNCG-435-3 cells were 3.2-fold less sensitive
than that of the docetaxel-treated parental or vector-transfected control cells (Fig. 1A).
To address whether anti-apoptosis was involved in SNCG-mediated resistance to docetaxel, we
examined the presence of cleaved poly (ADP-ribose) polymerase (PARP) as an apoptotic cell death marker.
Western analysis showed a significant increase in cleaved PARP in parental MCF-7 cells treated with
docetaxel. As shown in Fig. 1B, we found that the full-size PARP protein (116 kDa) was cleaved to yield an
85-kDa fragment after treatment of cells with docetaxel. In SNCG expressing MCF-7 (MCF-S6) cells,
however, PARP cleavage was observed at the significant decreased levels upon treatment with docetaxel.
The effect of SNCG expression on docetaxel resistance was further demonstrated by inhibiting
endogenous SNCG expression in SKBR3 and MDA-MB-231 cells. SNCG siRNA significantly reduced
endogenous SNCG expression in SKBR3 (insert, Fig. 1C) and MDA-MB-231 (insert, Fig. 1D) cells. In non-
treated cells, there was no significant difference in apoptosis between siSNCG-infected and control siCtrl-
infected cells. Treatment of siCtrl-infected SKBR-3 cells with docetaxel led to a 28% apoptotic cells. This
docetaxel-mediated apoptosis was significantly increased in the SNCG knockdown cells resulting in a 63%
of apoptotic cells (Fig. 1C). For MDA-MB-231 cells, while docetaxel treatment resulted in 32% apoptotic
cells, treatment of SNCG knockdown MDA-MB-231 cells with docetaxel resulted in 56% apoptotic cells
(Fig. 1D).
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We determined IC50 in response to docetaxel in several breast cancer cell lines, which have been
genetically modified for SNCG expression. As shown in Table 1, overexpression of SNCG into MCF-7 and
MDA-MB-435 cells increased IC50 values 3.3.and 2.4 fold, respectively. We previously established stable
T47D derived AS-3 clone by knockdown endogenous SNCG expression[27] . Knockdown endogenous
SNCG in T47D cells reduced IC50 values from 68 nM to 29 nM. These data suggest that SNCG renders
resistance to docetaxel-induced apoptosis.
We also tested the effect of SNCG on AMD resistance using a different MT disruptor nocodazole.
Unlike docetaxel, which is a MT-stabilizing agent, nocodazole is a MT inhibitor. The similar anti-apoptotic
effect of SNCG was also observed in nocodazole-induced apoptosis in SNCG transfected MDA-MB-435
cells (Fig. 1E). These data suggest that SNCG has a broad effect on AMD resistance.
SNCG renders tumor resistance to docetaxel. To determine whether SNCG-mediated docetaxel
resistance could be administered in vivo tumor xenograft model, we studied the tumor growth of MDA-MB-
435 and MCF-7 and their SNCG-stable transfected cells in response to docetaxel. Treatment of MDA-MB-
435 and 435-neo-1 tumors resulted in >65% tumor growth inhibition. Consistent with apoptotic data in cell
culture, SNCG-435-3 tumors were resistant to the treatment with only 34% tumor growth inhibition (Fig.
1F). To test if SNCG-mediated tumor resistance to docetaxel in vivo also occurs through anti-apoptotic
effect, we used a TUNEL assay to compare apoptotic index in docetaxel-treated samples from SNCG-435-3
tumors vs. MDA-MB-435 and 435-neo-1 tumors (Fig. 1G). Few TUNEL-positive cells were detected in
MDA-MB-435 and 435-neo-1 tumors without treatment. However, treatment with docetaxel remarkably
increased TUNE-positive cells (MDA-MB-435: from 7 to 36% positive cells; MDA-435-neo1: from 5 to
42% positive cells). Although docetaxel also enhanced TUNEL-positive cells in the samples obtained from
SNCG-435-3 tumors, the increase was significnatly reduced (from 5% without docetaxel to 17% with
docetaxel).
For MCF-7 xenografts, the growth of MCF-S6 tumor was stimulated much more by E2 than parental
MCF-7 tumor, which is consistent with the chaperon function of SNCG on ERα transcriptional
activation[27][28]. .As expected, the growth MCF-7 xenograft was significantly inhibited by docetaxel. At
25 days following treatment, docetaxel inhibited E2-stimulated tumor growth by 58%. Although the tumor
growth of MCF-S6 cells was also inhibited by docetaxel, the magnitude of growth inhibition reduced with a
slight 28% growth inhibition (Fig. 1H). This SNCG-mediated tumor resistance to docetaxel is consistent
with its anti-apoptotic effect in tumor as judged by a significant reduction of TUNEL-positive cells from
34% in MCF-7 xenografts to 14% in MCF-S6 xenografts (Fig. 1I). These results indicate that expression of
SNCG significantly inhibited antitumor and anti-apoptotic effect of docetaxel in tumor xenograft model.
Molecular modeling of interaction between SNCG and BubR1. BubR1 is a well-defined guardian
of the mitotic spindle, initiating mitotic arrest in response to the lack of tension and/or chromosome
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alignment across the mitotic plate. BubR1 is a 120-kDa multi-domain protein, having a conserved N-
terminal region, a central non-conserved region and a C-terminal serine/threonine kinase domain. The C-
terminal kinase domain is involved in the phosphorylation of critical components of a mitotic checkpoint,
whereas N-terminal is involved in interaction with SAC component Cdc20. Previous studies conducted
through yeast two-hybrid screening revealed an interaction of SNCG with BubR1[30]. To gain more insight
into their interacting mechanism and to determine whether such interaction will cause the structural changes
of BubR1, a docking analysis was carried out to understand the interaction between SNCG and the
crystallographic structure of human BubR1. The result of HADDOCK study had given many clusters of
which the top ranked one was selected for final analysis. As shown in Fig. 2, the confirmation of SNCG with
the highest binding score among docking results is the C-terminal of SNCG (AA 106-127) and the N-
terminal TPR (tetratricopeptidelike folds) motifs 2 and 3 of BubR1. The C-terminal tail region of SNCG is
rich in aspartic and glutamic acid residues, and is responsible for the flexible nature of this region.
Furthermore, the tail region SNCG has a chaperone activity[34] similar to that of tubulin[35]. The C-terminal
tail region of SNCG was found to be placed near the GIG (146-148) motif of the BubR1. GIG motif was
found to be crucial for interactions with Cdc20 protein. This motif was also conserved in the yeast Mad3
protein, indicating its importance in the interaction with Cdc20[36][37]. The interaction studies reveal the
presence of strong hydrogen bond and hydrophobic interactions (Fig. 2A1-A2). Glu117 of SNCG was found
to be involved in H-Bond interactions with Tyr116, Gln110, Arg114 and Gln145 of BubR1, respectively.
Similarly, strong H-Bond interactions were also observed between Glu120, Glu121, Asp127, Glu110 of
SNCG with Arg114, Lys113, Ser117, Asn144, and Asn145 of BubR1, respectively. Strong hydrophobic
contacts were observed between SNCG and BubR1. Gly117 of SNCG makes hydrophobic contact with
Gly111 of BubR1. Similar kinds of interactions were also observed between Val118, Gln107, Glu120,
Glu121, Glu110, and Asp127 of SNCG with Gly111, Lys182, Gly148, Gly146, Val149, Glu112, Lys113,
and Arg114 of BubR1, respectively. Importantly, Gly146 and Gly148 of conserved GIG motif and Asn144,
Gln145 and Val149 of BubR1 were residues that were targeted to great extent by the residues of SNCG.
Interestingly, amino acids in this region of BubR1 show high sequence similarity with region1 of Mad3,
which is known to interact with Cdc20[37]. This finding supports the suggestion that both Cdc20 and SNCG
may interact with the same region of BubR1.
Structural changes induced by the interaction of SNCG on BubR1. Comparing the
superimposition of the native and the docked structures of BubR1, we found significant structural variations
in three regions with the root mean square deviation of about 1.331 Å (Fig. 2B). First, the displacement in
the position of the residues in the loop (Gln 145- Ala152) connecting the TPR motif 2 and 3 was observed
(Fig. 2C). The functionally conserved GIG (Gly 146- Gly148) motif is located within this region. Interaction
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of the residues Gln 145, Gly 146 and Gly148 of BubR1 with SNCG residues had imposed a considerable
difference in the position of the residues located in this region. Second, a structural transition and deviation
was observed in the loop (Gln110-Pro119), which connects the TPR motifs 1 and 2 of BubR1 (Fig. 2D). The
transition in the secondary structure (Coil to Helix) of residues Lys113-Trp116 of this loop was a
predominant change observed in the docked BubR1 complex. Third, a huge displacement in the position of
N-terminal α helix (Arg60-Glu65) and the loop (Ile 66- Pro 74) connecting the N-terminal α helix with TPR
motif 1 (Pro74- Ala108)) was observed in the docked BubR1 complex (Fig. 2E). Importantly, the presence of
two KEN boxes accounts for the binding of BubR1 with Cdc20, of which the first box is placed at the N-
terminal helix of BubR1[37][38]. Interaction of SNCG at this region and the subsequent structural deviation
might account for the observed functional differences of BubR1, which might affect its binding to other key
members in the SAC, such as Cdc20.
The molecular modeling study indicates that the C-terminal AA 106-127 of SNCG interacts with
BubR1. To confirm this specific C-terminal interaction with BubR1, we constructed a deletion mutant of
SNCG, which the C-terminal region of 106-127 was deleted. While the wild type SNCG (F-SNCG) was able
to bind to BubR1, C-terminal deleted SNCG (D-SNCG) failed to bind to BubR1 under the same conditions
(Fig. 3A). These data are consistent with the molecular modeling analysis, which indicate that the C-terminal
AA 106-127 of SNCG binds to BubR1. We also determined whether SNCG-induced drug resistance to
docetaxel is mediated by its C-terminal interaction with BubR1. While the F-SNCG transfected MCF-7 cells
(MCF-S6) were resistant to docetaxel treatment, two clones of D-SNCG transfected cells (SNCG-D2 and
SNCG-D6) failed to convey resistance to docetaxel treatment; the sensitivity of D-SNCG cells to docetaxel-
induced apoptosis is similar to that of parental MCF-7 cells and the control clone MCF-neo1 cells (Fig. 3B).
We determined the tumor growth of MCF-7, F-SNCG-stable transfected MCF-S6, and D-SNCG stable
transfected SNCG-D2 cells in response to docetaxel (Fig. 3C). Treatment of MCF-7 tumor resulted in >60%
tumor growth inhibition. As expected, MCF-S6 tumors were resistant to the treatment with only 29% tumor
growth inhibition. However, for the growth of SNCG-D2 tumors, docetaxel inhibited tumor growth by 61%.
These data suggest that SNCG-induced drug resistance to docetaxel is mediated by its C-terminal interaction
with BubR1.
SNCG compromises BubR1 function. Upon spindle damage, BubR1 is activated and the activated
BubR1 then functions synergistically with Mad2 to activate anaphase-promoting complex (APC) by binding
and inhibiting Cdc20. Our molecular modeling study indicate that both SNCG and Cdc20 interact with the
same GIG motif of BubR1; and such SNCG-BubR1 interaction might account for the functional differences
of BubR1, which prevents itself from binding and inhibiting Cdc20. To further investigate the mechanism
through which SNCG influence BubR1 function, we examined whether SNCG-BubR1 binding may interfere
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BubR1 interaction with other SAC proteins of Cdc20 and Mad2. We carried out Co-IP experiments to
determine whether SNCG compromises the physical interaction between BubR1 and Cdc20 and Mad2.
SNCG binds to BubR1 in MCF-S6 cells; and this SNCG-BubR1 binding inhibits the physical interaction of
BubR1 with Cdc20 and Mad2 in MCF-S6 cells that had been synchronized and treated with docetaxel before
IP with anti-Cdc20 and anti-Mad2 (Fig. 4A Upper). Similarly, when endogenous SNCG was reduced in
MDA-MB-231 cells by siRNA, the interactions between endogenous BubR1 and Cdc20 and Mad2 were
significantly increased (Fig. 4B). To confirm that the effects of SNCG on BubR1 function relays on its
specific C-terminal interaction, we analyzed the effect of D-SNCG mutant on physical interactions between
BubR1 and Cdc20. While the full-length SNCG was able to reduce the BubR1 interaction with Cdc20, D-
SNCG failed to affect the interactions between BubR1 and Cdc20 (Fig. 4C). These data are consistent with
the molecular modeling analysis, which indicate that the C-terminal of SNCG binds to BubR1, causes a
structure change of functionally conserved GIG motif, and thus attenuates the BubR1 interaction with other
key SAC proteins Cdc20.
The kinase activity of BubR1 is essential for checkpoint signaling. Recently, activated BubR1 was
shown to phosphorylate specific targets, including itself, and to induce mitotic cell death. We therefore
analyzed BubR1 activity after the induction of mitotic arrest with docetaxel in the presence or absence of
SNCG. When we examined the effect of SNCG on the ability of BubR1 to phosphorylate itself and histone
H3 in vivo, a decrease in SAC kinase activity was observed in SNCG-positive clone MCF-S6 cells, as
evidenced by reduced phosphorylation of BubR1 and phospho-histone H3 (Fig. 4D). To confirm that this
reduced phosphorylation is mediated by BubR1, we immunoprecipitated BubR1 from docetaxel-arrested
both control MCF-7 and MCF-S6 cells and determined the in vitro ability of precipitated BubR1 to
phosphorylate histone H1. As demonstrated in Fig. 4E, expression of SNCG in MCF-S6 cells significantly
reduced the kinase activity of BubR1 to phosphorylate histone H1. These data indicate that SNCG/BubR1
binding reduces BubR1’s kinase activity and thus attenuates mitotic cell death.
To confirm that the reduced BubR1 function due to its interaction with SNCG contributes to decreased
docetaxel-induced mitotic arrested cells, we overexpressed BubR1 in MCF7-neo1 and MCF7-S6 cells by
transient transfection of pCS2-BubR1. Fig. 4F shows that exogenous expression of BubR1 did not
significantly alter the responses to the docetaxel-induced mitotic arrest in MCF7-neo1 clone. In contrast,
enforced BubR1 overexpression in MCF7-S6 cells significantly reversed SNCG-mediated resistance to
docetaxel-induced mitotic arrest and increased the mitotic index. These data provide additional evidence that
links the impaired mitotic checkpoint function to the decreased BubR1 function by SNCG expression.
SNCG compromises mitotic checkpoint function. Prolonged mitotic arrest by microtubule disruption
triggers apoptosis in the cells with normal mitotic checkpoint functions. Disruption of BubR1 function, e.g.
by SNCG, compromises checkpoint function, and thus lead to a reduced mitotic arrest. Antagonism of
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docetaxel cytotoxicity by SNCG is likely to result in compromising checkpoint function and a reduced
mitotic arrest in response to the drug. We determined mitotic indices of several breast cancer cell lines,
which have been alternated for exogenous expression of SNCG or genetically knockdown endogenous
SNCG. In MDA-MB-435 and control-transfected neo-435-1 cells, over 57% of the entire population was in
the mitotic stage following 20 h of docetaxel treatment. This compared to just 3.3% mitotic cells following
vehicle treatment (Fig. 5A). In contrast, forced expression of SNCG in SNCG-435-3 cells significantly
reduced the percentage of mitotic cells to <13% after docetaxel treatment. The effect of SNCG on anti-
mitotic arrest was also observed in SNCG stably transfected MCF-7 cells (Fig. 5B). Under the same
treatment condition, <10% of MCF-S6 cells were arrested at the mitotic phase whereas cells that do not
express SNCG (MCF-7 and control vector transfected MCF-neo1) cells were predominantly in the mitotic
stages (Fig. 5B). The effect of SNCG expression on mitotic arrest in response to docetaxel treatment was
further demonstrated by inhibiting endogenous SNCG expression in SKBR3 and MDA-MB-231 cells.
Docetaxel treatment caused a significant increase in mitotic arrest in SNCG knockdown MDA-MB-231 (Fig.
5C) and SKBR3 (Fig. 5D) cells. In contrast, expression of endogenous SNCG partially prevented docetaxel-
induced mitotic arrest. These results together clearly demonstrate that the normal mitotic checkpoint function
which is required for cells to be arrested at the mitotic phase by anti-microtubule agents is generally impaired
in SNCG expressing cells.
SNCG predicts docetaxel resistance in neoadjuvant treatment. Since SNCG compromsies BubR1
and SAC function, overrides docetaxel-induced mitotic checkpoint function, and renders resistance to
docetaxel-mediated apoptosis both in cells and in tumor xenografts, we determined the clinical relevance of
SNCG expression in predicting patient response to docetaxel-based therapy in a neoadjuvant trial, which
consists three cycles of standard neoadjuvant TE treatment (Docetaxel 75 mg/m2 1hr infusion and epirubine
60 mg/m2 IV). In this pilot trial, 30 patients were chosen for enrollment, and among them 60% (18/30) cases
were positive for SNCG expression. SNCG expression was analyzed by immunohistochemistry and defined
as percentage of tumor cells showing immunoreactivity as follows: SNCG-, <10%; SNCG+, 10-20%;
SNCG++, 20-50%; SNCG+++, 50-75%. Fig. 6A,B shows representatives of SNCG- and SNCG+++ tumors.
Due to the limited patient number and for the purpose of statistical analysis, 18 patients had ≥ SNCG++
tumors and were regarded as SNCG-positive and 12 patients had < SNCG++ tumors and were regarded as
SNCG-negative. None of these patients had received prior therapy for cancer. The age of patients was not
significantly different in SNCG+ group (51.56±9.61cm) vs. SNCG- group (53.42±10.90cm), p=0.626.
According to WHO Response Evaluation Criteria, 18 patients achieved partial response (PR), and among
them, 11 patients with PR were in SNCG- group and 7 in SNCG+ group. Twelve patients showed stable
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disease (SD) and no patients had progressive disease (PD). Although the sizes of the tumors were reduced
significantly after 3-cycle of therapy in both SNCG+ and SNCG- groups, for patients in the SNCG+ groups,
tumor sizes were reduced moderately (5.94 cm vs. 4.17 cm, p = 0.00); the tumor size reduction in the SNCG-
group was reduced more greatly (6.17 cm vs. 3.00, p = 0.000). The average tumor size in SNCG- group was
significantly smaller than that in SNCG+ group (Table 2).
There were no significant differences in expression of ER, PR, p53, and Her2 before and after the
chemotherapy in both groups. Consistent with the observed tumor regression data, the proliferation rate
(Ki67 expression) was significantly reduced in SNCG- group (p=0.001) after chemotherapy, but not in the
SNCG+ group (p=0.052). We also analyzed the apoptosis index. The apoptosis in SNCG- group was
significantly induced with a median increase from 11.83% to 25.92% (p = 0.021). However, apoptosis was
only increased slightly but not significant in SNCG+ tumors (8.17% vs. 10.56%, p = 0.411). Although
pretreatment apoptotic index was lower for SNCG+ positive tumors (8.17%) versus SNCG- tumors (11.83%),
this was not statistically significant. The average apoptotic index in SNCG- group was significantly higher
than that in SNCG+ group. Fig. 6C shows the changes of apoptotic index of individual tumors before and
after chemotherapy; among them there were two tumors with apoptotic index increased over 30% post
chemotherapy in the SNCG- group.
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Discussion.
Antimicrotubule drugs, e.g. docetaxel, the most widely prescribed drug class in oncology, are a
mainstay of metastatic cancer treatment during adjuvant therapy and to prevent metastases in post-surgery
cancer patients. Antimicrotubule drugs disrupt cellular microtubule networks and are thought to induce
mitotic catastrophe, which activates SAC, resulting in cells arrested in the mitotic phase and leading to
apoptosis. If SAC is compromised in cancer cells, the cells will escape the mitotic arrest without undergoing
apoptosis. From four different lines of investigation, we provide conclusive experimental evidence indicate
that SNCG renders a resistance to docetaxel treatment. First, we demonstrated that exogenous expression of
SNCG in MCF-7 or MDA-MB-435 cells markedly decreased docetaxel-induced apoptosis as compared to
their respective control neo clones. Conversely, knocking down the endogenous expression of SNCG in
SKBR3, T47D, and MDA-MB-231 cells increased apoptosis in response to docetaxel treatment. Second,
tumor xenograft studies further demonstrate that stable SNCG transfected MDA-MB-435 and MCF-7 cells
were less sensitive to docetaxel-induced antitumor effect as compared to SNCG-negative parental and their
control neo cells. Third, expression of SNCG in the patients with primary breast cancer indicates a poor
response to docetaxel-based chemotherapy in a pilot neoadjuvant trial. Forth, expression of SNCG inhibits
SAC function and mitotic arrest. While expression of SNCG prevents docetaxel-induced mitotic arrest,
knockdown endogenous SNCG enhances the mitotic arrest in response to docetaxel treatment.
Since SNCG expression overrides the mitotic checkpoint control and confers the cellular resistance to
antimicrotubule drug-caused apoptosis, this suggest that the normal mitotic checkpoint function which is
required for cells to be arrested at the mitotic phase and the sequent apoptosis by docetaxel is generally
impaired in SNCG expressing cells. Several key proteins for SAC function have been identified, which
include BubR1, Mad2, Centromere-associated protein-E (CENP-E), Cdc20, and others[39]. BubR1 kinase is
a key regulator of the SAC. The importance of BubR1 in the SAC is reflected by the observation that BubR1
forms the mitotic checkpoint complex (MCC) composed of Mad2, Bub3, BubR1, and Cdc20 and inactivates
the APC/C–Cdc20 complex[40]. Insufficient BubR1 function may induce a perturbed SAC function, and
thus leads to the misaligned chromosomes and high aneuploidy. A complete lack of SAC is incompatible
with viability in higher eukaryotes. However, the SAC is not “all or none.” It can be compromised to certain
degrees as indicated by the fact that the mice heterozygous for the essential components of SAC, e.g., BubR1
or Cdc20, are viable despite clear defects in the checkpoint function[33] . Studies have shown that disruption
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of BubR1 activity results in a loss of checkpoint control, chromosomal instability, and/or early onset of
malignancy[41]. Previous studies using yeast two-hybrid screening revealed an interaction of SNCG with
BubR1[30]. Since SNCG compromise SAC function and renders a resistance to antimicrotubule drug, we
reason that SNCG-BubR1 interaction may contribute to the inhibition of SAC as a result of nonmutational
inactivation. To study the molecular mechanism through which SNCG interacts with BubR1, we performed
docking analysis of the interaction between SNCG and BubR1. BubR1 is an essential mitotic checkpoint
protein with at least two functions: as an active kinase at unattached kinetochores and as a cytosolic inhibitor
of APC/C–Cdc20 complex. It is established that N-terminal Cdc20 binding domain of BubR1 is essential for
all of these functions[37]. We provide evidence that the C-terminal of SNCG binds directly to the N-terminal
TPR motifs 2 and 3 of BubR1, which is critical for binding to Cdc20[42]. The chaperone-like activity of
SNCG has been demonstrated in the cell-free system by assaying the aggregation of thermally denatured
proteins[43] . All three members in synuclein family share an extensive sequence homology. The most
conserved regions are in the N-terminal portion of the protein, In contrast, the C-terminal region (AA86-127)
of SNCG is quite different from synuclein α and β. A unique feature of chaperone proteins is a flexible
hydrophilic tail region in the C-terminal. Nuclear magnetic resonance spectroscopy has revealed that proteins
with chaperone-like activities have unstructured flexible solvent-exposed C-terminal extensions[44]. .The
polar and flexible C-terminal tail is thought to promote the interaction of the chaperone with the hydrophobic
region in the denatured protein and thus play a critical role in substrate-chaperone association. Indeed,
previous reports indicated that the C-terminus of SNCG is particularly important in performing the protein
binding and chaperone function[34][44]. Consistent with the molecular modeling analysis, our studies using
SNCG deletion mutants indicate that the C-terminal AA 106-127 is responsible for its specific binding to
BubR1 and such binding is able to reduce the BubR1 interaction with Cdc20. Furthermore, we also
demonstrated that C-terminal of SNCG is required to mediate drug resistance to docetaxel.
Since SNCG specifically interacts with the N-terminal Cdc20 binding domain of BubR1, which is
essential for its kinase activity and inhibition of Cdc20, we reason that SNCG expression may compromise
the mitotic checkpoint control by inhibiting the normal function of BubR1. We hypothesize that expression
of SNCG leads to a weakened SAC activation; this compromised SAC response is mediated by at least two
categories: 1) binding to and inhibiting BubR1 kinase activity; and 2) SNCG-BubR1 interaction may
attenuate or prevent the physical interaction of BubR1 with other key factors in SAC such as Mad2 and
Cdc20 in a competitive manner. Both Mad2 and BubR1 bind directly to Cdc20 and inhibit APC/C activity in
vitro [40]. However, combined BubR1 and Mad2 are much more potent APC/CCdc20 inhibitors than the
individual proteins[ 45 ]. This, together with the discovery of an in vivo mitotic checkpoint complex
consisting of Mad2, BubR1, Bub3 and Cdc20 and the demonstration that this complex strongly inhibits
APC/C activity in vitro[40] led to speculation that BubR1-Mad2-Cdc20 complexes to form the ultimate
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cytosolic APC/C inhibitor[37]. Mad2 and BubR1 both regulate the timing of mitosis in a kinetochore-
independent fashion. It has been proposed that this timing function requires BubR1 binding to Cdc20 and
Mad2 at the onset of mitosis when checkpoint proteins have yet to assemble[46]. Consistent with the
molecular modeling study, we demonstrated that SNCG-BubR1 interaction inhibits BubR1 phosphorylation,
the kinase activity, and the ability to bind to Cdc20 and Mad2. These studies suggest that SNCG expression
compromises the mitotic checkpoint control by inhibiting the normal function of BubR1. As the action of
antimicrotubule drugs rely heavily on the normal function of mitotic checkpoint machinery, in which BubR1
is a critical component, one of the key mechanisms underlying antimicrotubule drug resistance involves
acquired inactivation of SAC. In this regard, SNCG binds to the N-terminal TPR motif of BubR1, inhibits
BubR1 activity, prevents the formation of BubR1-Mad2-Cdc20 complexes, and thus results in an insufficient
of BubR1-related SAC function. Cancer cells, in the presence of SNCG, have a reduced ability to initiate and
maintain proper mitotic arrest, which leads to antimicrotubule drug resistance by premature exit, before cells
initiate apoptosis. The inhibitory effect of SNCG on BubR1 function may explain the induced resistance
against antimicrotubule drugs in breast[47], prostrate[48] , and lung[49] cancers. However, the relationship
between antimicrotubule drug resistance and SNCG is likely to be complex. Cancer cells resistant to
antimicrotubule drugs may have alternations in tubulin dynamics. In this regard, SNCG may act as a
functional microtubule associated protein, affect microtubule properties, and thus may lead to
antimicrotubule drug resistance.
Currently there are no clinically verified factors that can be used to predict docetaxel resistance. These
studies suggest that SNCG can be used as biomarker for docetaxel resistance. Interestingly, although SNCG
gene does not have a signal peptide, suggesting it is not a secreted protein, a secreted form SNCG can be
detected in sera of pancreatic[22] and colon[20] cancer and urine samples of bladder cancer[23]. In these
studies, SNCG protein is abnormally detected in a high percentage in sera[20][22] or urine samples[23] of
cancer patients but rarely expressed in healthy controls. Identification of cellular factors, particularly a
soluble serum factor, that are associated with the sensitivity to docetaxel-based treatment and development of
a predictive/prognostic marker to identify responders and non-responders would have great clinical
implications. The possibility of predicting that patients may not respond to docetaxel by measuring a single
serum biomarker might be quite compelling. Biomarker analysis is rarely simple or restricted to one protein.
It might be necessary to put SNCG expression in context with other possible biomarkers. Nonetheless, our
study will potentially lead to a new molecular profile of the tumor for the optimal patient selection for
antimicrotubule drugs and a new strategy of combining SNCG targeting with docetaxel as a novel
advantageous approach for treatment of cancer.
ACKNOWLEDGMENTS
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This study was supported in part by grants 81230054 and 91029739 from State Key Program of National
Natural Science Foundation of China; a grant from National Key Technology R&D Program for the 12th
Five-year Plan of china (2013BAI01B06); and a grant from State Key Laboratory of Reproductive Medicine.
This study was also supported in part by a grant 81102016 from National Natural Science Foundation of
China.
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[35] Guha S, Manna TK, Das KP, Bhattacharyya B. Chaperone-like activity of tubulin. The Journal of biological chemistry 1998; 273(46): 30077-80. [36] D'Arcy S, Davies OR, Blundell TL, Bolanos-Garcia VM. Defining the molecular basis of BubR1 kinetochore interactions and APC/C-CDC20 inhibition. The Journal of biological chemistry 2010; 285(19): 14764-76. [37] Malureanu LA, Jeganathan KB, Hamada M, Wasilewski L, Davenport J, van Deursen JM. BubR1 N terminus acts as a soluble inhibitor of cyclin B degradation by APC/C(Cdc20) in interphase. Developmental cell 2009; 16(1): 118-31. [38] Burton JL, Solomon MJ. Mad3p, a pseudosubstrate inhibitor of APCCdc20 in the spindle assembly checkpoint. Genes & development 2007; 21(6): 655-67. [39] Doncic A, Ben-Jacob E, Barkai N. Evaluating putative mechanisms of the mitotic spindle checkpoint. Proceedings of the National Academy of Sciences of the United States of America 2005; 102(18): 6332-7. [40] Sudakin V, Chan GK, Yen TJ. Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2. The Journal of cell biology 2001; 154(5): 925-36. [41] Dai W, Wang Q, Liu T, Swamy M, Fang Y, Xie S, et al. Slippage of mitotic arrest and enhanced tumor development in mice with BubR1 haploinsufficiency. Cancer research 2004; 64(2): 440-5. [42] Davenport J, Harris LD, Goorha R. Spindle checkpoint function requires Mad2-dependent Cdc20 binding to the Mad3 homology domain of BubR1. Experimental cell research 2006; 312(10): 1831-42. [43] Souza JM, Giasson BI, Lee VM, Ischiropoulos H. Chaperone-like activity of synucleins. FEBS letters 2000; 474(1): 116-9. [44] Smulders R, Carver JA, Lindner RA, van Boekel MA, Bloemendal H, de Jong WW. Immobilization of the C-terminal extension of bovine alphaA-crystallin reduces chaperone-like activity. The Journal of biological chemistry 1996; 271(46): 29060-6. [45] Fang G. Checkpoint protein BubR1 acts synergistically with Mad2 to inhibit anaphase-promoting complex. Molecular biology of the cell 2002; 13(3): 755-66. [46] Meraldi P, Draviam VM, Sorger PK. Timing and checkpoints in the regulation of mitotic progression. Developmental cell 2004; 7(1): 45-60. [47] Sudo T, Nitta M, Saya H, Ueno NT. Dependence of paclitaxel sensitivity on a functional spindle assembly checkpoint. Cancer research 2004; 64(7): 2502-8. [48] Lanzi C, Cassinelli G, Cuccuru G, Supino R, Zuco V, Ferlini C, et al. Cell cycle checkpoint efficiency and cellular response to paclitaxel in prostate cancer cells. The Prostate 2001; 48(4): 254-64. [49] Goncalves A, Braguer D, Kamath K, Martello L, Briand C, Horwitz S, et al. Resistance to Taxol in lung cancer cells associated with increased microtubule dynamics. Proceedings of the National Academy of Sciences of the United States of America 2001; 98(20): 11737-42.
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Tables Table 1. Cytotoxicity of docetaxel.
Cell lines SNCG expression status Cytotoxicity IC50
MCF-7 negative 27
MCF-S6 transfected 89
MDA-MB-435 negative 63
SNCG-435-3 transfected 153
T47D positive 68
AS-3 knockdown 29
Stable SNCG transfected MCF-7 (MCF-S6), MDA-MB-435 (SNCG-435-3), and SNCG knockdown T47D (AS-3) cells were
previously established. All six cell lines were treated with docetaxel (ranging from 10 nM to 250 nM) for 24 h. Cytotoxicity was
measured by MTT assay. The IC50 values were defined as the concentration of drug eliciting 50% cell killing and determined by
regression analysis.
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Table 2. Changes of tumor size, Ki67 expression and apoptotic index
pre-chemotherapy post-chemotherapy t P value
Size (cm)
SNCG+ 5.94±1.76 4.17±1.10 5.97 0.000
SNCG- 6.17±1.64 3.00±0.95 7.82 0.000
t -0.347 3.000
P value 0.731 0.006
Ki67 (%)
SNCG+ 26.67±18.55 20.56±11.49 0.09 0.052
SNCG- 32.50±14.85 14.17±9.73 4.26 0.001
t -0.910 1.582
P value 0.370 0.125
Apoptotic Index(%)
SNCG+ 8.17±6.64 10.56±8.12 -0.84 0.411
SNCG- 11.83±8.79 25.92±19.62 -2.69 0.021
t -1.301 -2.569
P value 0.204 0.023
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Figure Legends
Fig. 1. SNCG renders a drug resistance. A, effect of SNCG on docetaxel resistance in breast cancer cells.
SNCG-negative MCF-7 and MCF-neo1 (vector transfect clone), SNCG stably transfected MCF-S2 and
MCF-S6 cells (Upper); SNCG-negative MDA-MB-435 and neo-435-1 (vector transfected clone), and SNCG
stably transfected SNCG-435-3 cells (Bottom) were treated with or without 100 nM docetaxel for 2 days and
apoptotic cells were determined. The numbers represent means ± SD of three cultures. Statistical
comparisons of treated vs. control indicate p < 0.001. B, effects on apoptotic biomarkers. MCF-7 and MCF-
S6 cells were treated with or without docetaxel (100 nM, 48 h). Equal amount of total cellular protein was
subjected to Western analyses of PARP, cleaved PARP, and actin. C-D, effects of knockdown SNCG
expression on SKBR-3 (C) and MDA-MB-231 (D) cells on docetaxel resistance. Inserts indicate the reduced
SNCG expression in the siRNA knockdown cells. Cells were treated and analyzed as described for MCF-7
and MDA-MB-435 cells. E, effects of SNCG on nocodazole resistance. MDA-MB-435 and SNCG-435-3
cells were treated with nocodazole (0.5 μM, 30 hours) followed by analysis of apoptosis. F and H, SNCG
renders tumor resistance to docetaxel. Tumor growths of MDA-MB-435 (F) and MCF-7 (H) xenografts in
response to docetaxel treatment. Tumor cell injection and estrogen supplement (for MCF-7 cells) were
described in Materials and Methods. Mice bearing established tumors were treated with either vehicle control
or docetaxel (25 mg/kg, i.p., 1 time/5-days for 25 days). All mice were sacrificed at day 25 following the
first drug treatment. Statistical comparison for tumor sizes in docetaxel treated SNCG-negative MDA-MD-
435 and 435-neo-1 mice relative to mice in other groups indicates *P <0.01. Statistical comparison for tumor
sizes in docetaxel treated MCF-7 mice relative to mice in other groups indicates *P <0.005. G and I, effects
of SNCG on apoptosis of MDA-MB-435 (G) and MCF-7 (I) tumor xenografts. Histological sections of
tumors from docetaxel-treated or control mice were analyzed for apoptosis (TUNEL assay). Statistic analysis
showed that docetaxel-treated SNCG-expressing tumor tissues had a significantly lower percentage of
TUNEL-positive cells than did docetaxel-treated SNCG-negative tumor tissues (P <0.01).
Fig. 2. Structure analyses of the in silico interaction between BubR1 with C-terminal region of SNCG. A.
Docked interacting conformation of BubR1 (determined by x ray crystallography) with the in silico predicted
structure of SNCG-C terminal region (color code: blue). A1-A2. The interaction mode between the residues
of BubR1 (color codes: yellow) involved in binding with SNCG (color code: sky blue), which influence the
displacement in the GIG motif-1 (A1) and those that cause the structural transition 1 (A2). B. Structural
superimposition of native BubR1 x-ray crystallographic structure [color code: green] with the structure
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obtained after docking with C-terminal region of SNCG (color code: chocolate brown). C. The displacement
in the position of GIG motif of BubR1 (color code: green) compared with that of native BubR1 structure
(color code: red); D. The structural change occurred in the BubR1 [Gln110-Pro119] (color code: blue)
compared with that of native BubR1 structure (color code: green). E. The structural displacement occurred in
the region [Pro74- Ala108] of BubR1 (color code: violet purple) with native BubR1 structure (color code:
lemon green)
Fig. 3. Effects of SNCG C-terminal on interaction with BubR1 and drug resistance. MCF-7 cells, engineered
to express either full lenght F-SNCG or C-terminal deleted D-SNCG (AA 106-127 deleted), were slected for
stable clones. A, effects on interaction with BubR1. F-SNCG and D-SNCG transfected MCF-7 cells were
treated with docetaxel (80 nM, 20 h) and extracts were immunoprecipitated with polyclonal SNCG antibody
and blotted with BubR1 antibody. Total cellular (before IP) proteins of SNCG, BubR1, and actin were also
analyzed as inputs. B, in vitro effect of SNCG on docetaxel resistance. SNCG-negative parental MCF-7 and
neo transfected control MCF-neo1 cells (vector transfected clone), F-SNCG stably transfected MCF-S6 cells,
and D-SNCG stably trnasfected MCF-D2 and MCF-D6 cells were treated with or without 100 nM docetaxel
for 2 days and apoptotic cells were determined. The numbers represent means ± SD of three cultures.
Statistical comparisons of treated vs. control indicate p < 0.001. C, in vivo effect of SNCG on docetaxel
resistance in tumor xenograft model. Tumor cell injection and estrogen supplement were described in
Materials and Methods. Mice bearing established MCF-7, MCF-S6, and MCF-D2 tumors were treated with
either vehicle control or docetaxel (25 mg/kg, i.p., 1 time/5-days for 25 days). All mice were sacrificed at
day 25 following the first drug treatment. Statistical comparison for tumor sizes in docetaxel treated SNCG-
negative MCF-7 and D-SNCG stably transfected MCF-D2 mice relative to mice in other groups indicates *P
<0.01.
Fig. 4. SNCG binds to BubR1, compromises BubR1 interaction with Cdc20 and Mad2, and inhibits BubR1
kinase activity. A-B, effects of SNCG on BubR1’s interaction with Cdc20 and Mad2 in MCF-7 (A) and
MDA-MB-231 (B) cells. MCF-7 (parental and MCF-S6) and MDA-MB-231 (control siRNA and SNCG
siRNA) cells were treated with docetaxel (80 nM, 20 h) and extracts were immunoprecipitated with SNCG,
Cdc20, and Mad2 and blotted with BubR1 antibody. Total cellular (before IP) proteins of BubR1, SNCG,
Cdc20, and actin were also analyzed as inputs. C, MCF-7 cells, engineered to express full lenght F-SNCG
and C-terminal deleted D-SNCG (AA 106-127 deleted), were treated with docetaxel (80 nM, 20 h) and
extracts were immunoprecipitated with BubR1 and blotted with Cdc20 antibody. D, expression of SNCG in
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MCF-7 cells correlates with reduced SAC kinase activity. Levels of phospho-BubR1 (P) and phospho-
histone H3 (p-his3) (representing SAC kinase activity in vivo) were determined by Western blotting on
extracts from MCF-7 and MCF-S6 cells treated with or without docetaxel (80 nM. 20 h). E, SNCG inhibits
BubR1 kinase activity in vitro. MCF-7 and MCF-S6 cells were treated with 80 nM docetaxel for 20 h.
Endogenous BubR1 was immunoprecipitated from docetaxel-arrested cells and immunoblotted with anti-
BubR1 antibody or kinase activity assayed after addition of histone H1 and [32P]ATP. F, overexpression of
BubR1 restored the mitotic checkpoint control. MCF-neo1 and MCF-S6 cells were transfected with pCS2-
BubR1 or control vector. At 30 h post-transfection, cells were treated with DMSO or 80 nM docetaxel for 20
h. For each cell line, 200-300 cells randomly chosen from 5 different views were scored for mitotic arrest
based on MPM-2 immuno-reactivity. We examined the status of phosphoproteins that are specifically
recognized by MPM-2 antibody and found only in mitotic phase cells using flow cytometr. Data are
expressed as percent of MPM-2-reactive cells in the propidium iodide (PI)-positive population. The numbers
represent means ± SD of three cultures. A representative experiment is shown.
Fig. 5. SNCG renders lower mitotic index. Cells from MCF-7 (A), MDA-MB-435 (B), SKBR3 (C), and
MDA-MB-231 (D) were cultured in cover slips inserted into wells of 24-well culture plates and treated with
DMSO or 80 nM docetaxel for 20 h. Data are expressed as percent of MPM-2-reactive cells in the propidium
iodide (PI)-positive population as described in Fig. 4. The numbers represent means ± SD of three cultures.
Fig 6. A-B, representative SNCG immunohistochemical stainings for SNCG- and SNCG+++ tumors.
Sections were immunohistochemically stained with specific anti-SNCG antibody with brown color
indicating SNCG protein expression in breast cancer cells. All sections were also counterstained lightly with
hematoxylin for viewing non-SNCG-stained cells. A serial slide from the same block was also incubated
with nonimmunized control IgG, and no detectable background staining was observed. C, individual changes
in apoptotic index at pre-treatment and post-treatment of docetaxel and epirubine for patients with SNCG-
negative and -positive tumors.
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Published OnlineFirst January 15, 2014.Mol Cancer Ther Shuyu Miao, Kejin Wu, Bo Zhang, et al. and renders resistance to antimicrotubule drugSynuclein gamma compromise spindle assembly checkpoint
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