Suyu Miao1 2 1 Mingjie Zhu3 Yunshu Lu2 Ramadas Krishna4€¦ · 15/01/2014 · 1 Synuclein gamma...

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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

on January 31, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on January 15, 2014; DOI: 10.1158/1535-7163.MCT-13-0671

http://mct.aacrjournals.org/

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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




12

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




13

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




14

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.




15

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




16

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




17

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




18

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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21

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.




22

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




23

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




24

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.




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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Suyu Miao1 2 1 Mingjie Zhu3 Yunshu Lu2 Ramadas Krishna4€¦ · 15/01/2014 · 1 Synuclein gamma...

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