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Dermatan sulfate epimerase 1 expression and mislocalization may interfere with dermatan sulfate synthesis and breast cancer cell growth
Journal Pre-proof Dermatan sulfate epimerase 1 expression and mislocalization may interfere with dermatan sulfate synthesis and breast cancer cell growth Eduardo Listik, Everton Galvão Xavier, Maria Aparecida da Silva Pinhal, Leny Toma PII:
S0008-6215(19)30581-6
DOI:
https://doi.org/10.1016/j.carres.2020.107906
Reference:
CAR 107906
To appear in:
Carbohydrate Research
Received Date: 3 October 2019 Revised Date:
10 December 2019
Accepted Date: 3 January 2020
Please cite this article as: E. Listik, Everton.Galvã. Xavier, M.A.d. Silva Pinhal, L. Toma, Dermatan sulfate epimerase 1 expression and mislocalization may interfere with dermatan sulfate synthesis and breast cancer cell growth, Carbohydrate Research (2020), doi: https://doi.org/10.1016/ j.carres.2020.107906. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
1
Dermatan sulfate epimerase 1 expression and mislocalization may interfere with dermatan sulfate synthesis and breast cancer cell growth Eduardo Listik#, Everton Galvão Xavier#, Maria Aparecida da Silva Pinhal & Leny Toma*
Affiliation: Department of Biochemistry, Universidade Federal de São Paulo, Rua Três de Maio, 100, São Paulo, SP, Brazil. CEP: 04044-020. [email protected]; [email protected]; [email protected]; [email protected].
Corresponding author: Leny Toma, Ph.D. Department of Biochemistry Universidade Federal de São Paulo Rua Três de Maio, 100, 5th floor, CEP: 04044-020 São Paulo, SP, Brazil. Phone: +55-11-5573-6407 Email: [email protected]
#
Authors share equal contribution.
2 1
ABSTRACT
2
Dermatan sulfate (DS) is a glycosaminoglycan (GAG) that is produced through the
3
epimerization of the glucuronic acid on chondroitin sulfate into iduronic acid (IduA) by
4
dermatan sulfate epimerase (DS-epi) 1 and 2. Proteoglycans (PGs) play essential physiological
5
and pathological roles during cellular development, proliferation, differentiation, and cancer
6
metastasis. DS proteoglycans play vital roles during the process of tumorigenesis, due to the
7
increased flexibility of the polysaccharide chain in the presence of IduA residues, which
8
facilitate specific interactions with proteins, such as growth factors, cytokines, and angiogenic
9
factors. Furthermore, DS-epi is highly expressed in many tumors, especially in esophageal
10
squamous cell carcinoma. This study aimed to investigate the expression of DS-epi1 in multiple
11
breast cancer cell lines, including MCF7 (luminal A), MDA-MB-231 (triple-negative) and
12
SKBR3 (human epidermal growth factor receptor 2-positive), and its involvement in cancer
13
progression. A SKBR3 variant, SKBR3m, presented the most erratic cell growth pattern when
14
compared with those for MCF7 and MDA-MB-231. Moreover, SKBR3m cells demonstrated
15
the highest level of DS-epi1 gene expression and higher
16
protein level, MCF7 cells displayed the highest protein level for DS-epi1, whereas MDA-MB-
17
231 cells had the lowest level. DS-epi1 was found in vesicles and in the perinuclear
18
compartment only in SKBR3m cells, suggesting localization in the Golgi apparatus in these
19
cells, in contrast with the cytoplasmic localization observed in MCF7 and MDA-MB-231 cells.
20
The cytoplasm location of DS-epi1 likely compromised the formation of DS chains, but the
21
core protein was detected using a decorin antibody. Golgi-specific labeling confirmed the
22
localization of DS-epi1 in SKBR3m cells at the Golgi apparatus, indicating that the location of
23
the enzyme was a determinant for the synthesis of DS in this cell line, suggesting that DS may
24
play a decisive role in the tumor growth observed in this breast cancer cell line.
35
S-DS content. However, at the
3 25
Keywords: Breast cancer; cancer biology; dermatan sulfate; decorin proteoglycan;
26
Golgi apparatus.
27
1 INTRODUCTION
28
Breast cancer is one of the most prevalent types of tumors among women worldwide,
29
with more than 1.7 million new registered cases and 522,000 deaths every year. The incidence
30
of breast cancer has continued to grow, and more than 3.2 million new cases are expected by
31
2050 [1]. Breast cancer pathology can be classified according to morphologic, histologic, and
32
molecular parameters. Morphologically, breast tumors can be categorized as infiltrating ductal
33
carcinomas of no particular type, infiltrating lobular and tubular carcinomas, mucinous
34
carcinomas, and many others. Histological classifications allow the sub-categorization of
35
breast tumors, using characteristics such as cellular differentiation, nuclear pleomorphism,
36
and mitotic counts. At the molecular level, breast tumors can be classified according to the
37
presence or absence of estrogen receptor (ER), progesterone receptor (PR), or human
38
epidermal growth factor receptor type 2 (HER2). The majority of breast tumors are ER-
39
positive; however, many subgroups can be characterized, such as luminal A and B tumors,
40
which are ER+/PR+ [2-4].
41
Proteoglycans (PGs) are macromolecules that possess a protein core, to which one or
42
more glycosaminoglycan (GAG) chains are covalently attached [5]. Such molecules have
43
essential biological functions during vascularization and cancer metastasis and assist many
44
signaling proteins in the extracellular matrix [6, 7]. During tumoral development and growth,
45
the expression of PGs is markedly modified, frequently upregulated, and many PGs influence
46
or inhibit cell proliferation, depending on the tissue type [8-10].
47
Chondroitin sulfate (CS) is a GAG that is naturally found in the extracellular matrix of
48
connective tissue, either on the cell surface or in intracellular compartments. A disaccharidic
4 49
unit, composed of D-glucuronic acid (GlcA, β-1,4) and N-acetyl-D-galactosamine (GalNAc,
50
β-1,3), can be found in CS [11, 12]. Dermatan sulfate (DS), in contrast, possesses the GlcA
51
epimer iduronic acid (IdoA). Both GAGs are structurally similar, and they appear
52
concomitantly in PGs [7, 13].
53
GAG biosynthesis begins at the rough endoplasmic reticulum (RER) and proceeds to
54
the Golgi apparatus. In the RER, GAG synthesis begins with the formation of a
55
tetrasaccharidic sequence of GlcA, β1→3, D-galactose (Gal), β1→3, Gal, β1→4, xylose (Xyl),
56
β1→ [14-16]. After the addition of xylose and D-galactose, the synthetic process is transferred
57
to the Golgi apparatus [17, 18]. The fifth added sugar determines whether the GAG will
58
become a CS/DS or heparan sulfate (HS)/heparin (Hep) [19, 20]. The extension of CS occurs
59
through the activity of the chondroitin synthase enzyme. CS chains in mammals are
60
commonly sulfated on C-4 or C-6 positions of GalNAc, generating the isomers chondroitin-4-
61
sulfate or chondroitin-6-sulfate, respectively [21-23]. GlcA/IdoA can also be sulfated on the
62
C-2 position, either in CS or DS [24, 25]. Epimerization proceeds sulfation, during which
63
dermatan epimerase (DS-epi) 1 and 2 act on the C-5 of GlcA to generate an IdoA moiety [26,
64
27].
65
PGs play significant roles in several physiological and pathological cellular processes.
66
The results obtained in the present study may indicate that higher expression levels of DS-
67
epi1 in cells derived from breast tumors could be linked with higher growth rates. However,
68
the cells that express higher levels of DS-epi1 do not show higher DS-epi1 protein levels;
69
instead, DS-epi1 presents with a more specific localization in vesicles. This result led us to
70
question whether the vesicle-localized DS-epi1 found in cells with higher levels of DS-epi1
71
expression is functional.
72
2 MATERIALS AND METHODS
5 73
2.1 Materials
74
Anti-HS-epi (polyclonal rabbit, IgG, NBP2-31872) and anti-DS-epi1 (polyclonal
75
rabbit, IgG, NBP1-87962) antibodies were purchased from Novus Biologicals (Centennial,
76
Colorado, USA). Anti-decorin (monoclonal mouse, IgG, MA5-29240) was purchased from
77
Thermo Fisher (Waltham, Massachusetts, USA). All secondary antibodies, including anti-
78
rabbit Alexa Fluor 488 (cat. A-21206), anti-mouse Alexa Fluor 633 (cat. A-21050),
79
phalloidin-647 (cat. A22287), and lectin HPA from Helix Pomatia-647 (cat. L32454), were
80
purchased from Thermo Fisher. RPMI1640, Dulbecco’s Modified Eagle Medium (DMEM),
81
and fetal bovine serum (FBS) were all obtained from Gibco® (from Thermo Fisher).
82
2.2 Cell culture
83
MDA-MB-231 (ATCC® HTB-26TM), MCF7 (ATCC® HTB-22TM) and SKBR3
84
(ATCC® HTB-30TM) cells were purchased from the American Type Culture Collection
85
(ATCC, Manassas, Virgínia, USA). MDA-MB-231 and MCF7 cells were cultivated in
86
DMEM, whereas SKBR3 cells were cultivated in RPMI1640. SKBR3 cells were used in
87
specific assays: SKBR3m from a high passage (> 50), and SKBR3a from low passage levels
88
(< 5), as it has been previously described that breast cancer cell lines may acquire a more
89
aggressive profile in higher passages [28-31]. All media were supplemented with 10% FBS
90
(v/v) and 1% penicillin/streptomycin (v/v) The cells were grown at 37 °C, in a humidified
91
atmosphere containing 5% CO2.
92
2.3 MTT assay
93
The MTT assay was performed using the tetrazolium salt (MTT) reagent, as
94
previously described [32]. In brief, MDA-MB-231, MCF7, and both types of SKBR3 cells
95
were plated at a 3×103 cell/mL density in a 96-well plate. A 5 mg/mL sterile solution of MTT
96
in phosphate-buffered saline (PBS) was prepared, and 10 µL was added 4 h prior to cell
6 97
incubation for 24, 48, and 72 h. In some assays, a 96-h time point was also analyzed. At each
98
time point, the supernatant was removed from each well, and the formazan precipitate was
99
solubilized with 120 µL dimethylsulfoxide (DMSO). The plate was then briefly agitated, and
100
the absorbance of each well was read using a plate reader at 570 nm. The optical density of
101
blank wells was subtracted from each experimental well, and the results were normalized
102
against those for control wells to which MTT was not added.
103
2.4
104
electrophoresis
35
S-Sulfate metabolic radiolabeling of GAGs and analysis by agarose gel
105
MDA-MB-231, MCF7 and both types of SKBR3 cells were cultivated in either 35-
106
mm dishes or 24-well plates until they reached 60% confluence. Then, they were incubated
107
with Na235SO4 (100 µCi/mL) for 24 h. The medium was replaced with OptiMEM during the
108
radioactive pulse.
109
The medium was collected, and cells were washed twice with 2 mL cold PBS and
110
collected by scraping in 500 µL 3.5 M urea buffer (pH 7.4). PGs in both the collected media
111
and the cell extracts were proteolyzed with proteinase K at a final concentration of 100
112
µg/mL, for 3 h at 40 °C, before being inactivated at 95 °C for 10 min. Alternatively, the PGs
113
from the cell extracts and media were precipitated with three volumes of ethanol, in the
114
presence of 200 µg CS as a carrier, and incubated overnight at -20 °C. The solution was
115
centrifugated at 3,000 rpm for 10 min, and the precipitate was left to dry at a vacuum
116
concentrator. A proteolytic solution, consisting of 0.9 mM alcalase in 100 mM Tris-HCl (pH
117
7.4), was added and incubated for two days at 54 °C, followed by inactivation by boiling for 2
118
h.
119
In some experiments, after proteolysis, GAGs were digested with either chondroitinase
120
AC (0.1 units in 20 mM Tris-HCl, pH 7.2, 2% glycerol), chondroitinase ABC (0.1 units in 50
7 121
mM Tris-HCl, 60 mM sodium acetate, 0.02% of BSA (v/v)), both from Sigma Aldrich (San
122
Luis, Missouri, USA) or water, as a control. All samples were incubated overnight at 37 °C
123
(for chondroitinases). Then, agarose electrophoresis was performed, as previously described
124
[33]. In brief, 5 µL of each sample were applied to agarose gel slabs in 0.05 M 1,3-
125
diaminepropane acetate, pH 9.0. Afterward, GAGs in the gel slabs were precipitated for 1 h
126
with 0.2% cetyltrimethylammonium bromide (v/v), and then air-dried and stained with a
127
solution composed of 0.1% toluidine blue, 50% ethanol, and 1% acetic acid. The gel was
128
destained in a solution composed of 50% ethanol and 1% acetic acid and exposed to a
129
phosphor screen for visualization and quantification using a Cyclone® PhosphorImager. The
130
data, in counts per minute (cpm), were obtained by comparison with a standard radioactive
131
ink measured using a liquid scintillation counter (TRI-CARB 4910TR, Perkin-Elmer,
132
Waltham, Massachusetts, USA). The final value was normalized against total cell protein
133
content.
134
2.5 RNA extraction and real-time quantitative reverse transcription PCR (RT-
135
qPCR)
136
MDA-MB-231, MCF7, and SKBR3m cells were cultivated in 60-mm dishes until 90%
137
confluence was achieved. The medium was removed, and cells were incubated for 5 min with
138
1 mL TRIzolTM reagent. The lysate solution was transferred to tubes and mixed with 500 µL
139
chloroform by inversion. The tubes were centrifuged for 30 min, at 12,000 rpm, at 4 °C, and
140
the aqueous phase was transferred to a new tube. RNA was precipitated with 500 µL
141
isopropanol and sequentially washed with 1 mL 75% ethanol. The RNA pellet was left to dry
142
in a vacuum concentrator and resuspended in 40 µL RNase-free water.
143
Total RNA was quantified in a Nanodrop (NanoVueTM Plus, GE Healthcare, Chicago,
144
Illinois, USA), and a sample containing 2 µg total RNA and 2 µM oligo(dT)15 primers in 5 µL
145
RNase-free water was incubated at 70 °C for 5 min and then stored at 4 °C to minimize the
8 146
formation of RNA secondary structures. A 15 µL mixture, containing 667 µM dNTP mix, 4
147
mM MgCl2, 10.7 U/µL Improm-II reverse transcriptase, Improm-II reverse transcriptase
148
buffer (1.33×) and 1.33 U/µL Ribolock RNAse Inhibitor (Thermo Fisher Scientific), was
149
added to each sample. The reverse transcription reaction conditions were as follows:
150
annealing for 5 min at 25 °C, extension at 42 °C for 60 min, and enzyme inactivation at 70 °C
151
for 10 min.
152
cDNA was also quantified, and a quantitative PCR (qPCR) reaction mixture was
153
prepared to contain 50 ng/mL cDNA, 500 nM forward primers, 500 nM reverse primers, and
154
5 µL SYBR® Green PCR Master Mix (Applied Biosystems by Thermo Fisher), in a total
155
volume of 20 µL. The primers used in this assay are listed in Table 1. The qPCR conditions
156
included an activation phase for 10 min at 95 °C, and 40 cycles of denaturation (15 s at
157
95 °C), and annealing (1 min at 60 °C), with data collection occurring during the last step.
158
Table 1. Primers used in RT-qPCR.
Gene
Forward and reverse primers (5’-NNN-3’)
F: 5’-TGTGTGCTGTATCCTGAGAACA-3’ Dermatan sulfate epimerase 1 R: 5’-CAAGGCGCATCTTTCACCAC-3’ F: 5’-TTCCAAAGTCTATGCACAGGAGC-3’ Heparan sulfate epimerase R: 5’-TCCACATTGTAGCCTTCAAAAGAC-3’ F: 5’-TGGACCGTTTCAACAGAGAGGCTT 3’ Decorin proteoglycan R: 5’-TGGACCACTCGAAGATGGCATTGA-3’ Biglycan proteoglycan
F: 5’-AAGCTCAACTACCTGCGCATCTCA-3’
9 R: 5’-ATCATCCTGATCTGGTTGTGGCCT-3’ F: 5’-TAGCACTGCCCTTGGAATTTGTGC-3’ Versican proteoglycan R: 5’-AGCGGAGACCAGTGTGAACTTGAT-3’ F: 5’-TCGACAGTCAGCCGCATCTTCTTT-3’ GAPDH R: 5’-ACCAAATCCGTTGACTCCGACCTT-3’
159
2.6 Flow cytometry staining and data collection
160
MDA-MB-231, MCF7, and SKBR3m cells were cultivated in 100-mm dishes until
161
90% confluence was reached. The cells were detached using a 20-min incubation with 500
162
µM ethylenediaminetetraacetic acid (EDTA). The cells were collected by centrifugation,
163
distributed into tubes, and fixed with 200 µL 2% formaldehyde in PBS for 20 min, on ice.
164
The cells were washed sequentially with PBS and 0.1 M glycine and then permeabilized with
165
100 µL 0.01% saponin in PBS for 30 min on ice. After washing with PBS, the cells were
166
incubated with 50 µL antibody solution at 1:100 dilution for 1 h 30 min (anti-DS-epi1, anti-
167
HS-epi, or anti-decorin), diluted in 1% BSA in PBS. After washing with PBS, 50 µL
168
secondary antibody solution was added, containing either anti-rabbit Alexa Fluor 488 (1:200)
169
or anti-mouse Alexa Fluor 633 (1:200), for 40 min. The cells were resuspended in 200 µL
170
PBS and counted with a BD FACSCalibur (BD Biosciences). The fluorescence geometric
171
mean average of the cell population was used to compare flow cytometry data among cell
172
lines, and histograms were plotted for visual assessments.
173
2.7 Immunofluorescence staining and confocal imaging
174
MDA-MB-231, MCF7, and SKBR3m cells were plated at a 2×105 cell/mL density on
175
sterile borosilicate coverslips in a 24-well plate and incubated overnight. The supernatant was
10 176
removed, and cells were washed with cold PBS, followed by fixation with 4%
177
paraformaldehyde that was pre-warmed to 37 °C for 15 min. The coverslips were washed
178
three times with PBS, one time with 0.1 M glycine, and once again with PBS, for 5 min each
179
wash. The cells were then blocked and permeabilized with a solution containing 0.1% Triton
180
X-100 and 3% BSA for 1 h. Anti-DS-epi1 (rabbit IgG, 1:100) or anti-HS-epi (rabbit IgG,
181
1:150) were diluted in 0.05% Triton X-100 with 1% BSA in PBS were added to the cells and
182
incubated overnight at 4 °C. The coverslips were washed three times with PBS for 10 min and
183
incubated with anti-rabbit Alexa Fluor 488 (1:200) and phalloidin-647 (1:200) in 0.05%
184
Triton X-100 with 1% BSA in PBS, for 1 h at room temperature. The cells were washed three
185
times with PBS for 5 min and incubated with 4′,6-Diamidine-2′-phenylindole dihydrochloride
186
(DAPI, 1:2,000 in PBS) for 15 min. The coverslips were washed five times with PBS for five
187
minutes, dipped twice in cold type I water, mounted with FluoromountTM onto clean slides,
188
and sealed with colorless nail polish. For Golgi apparatus staining, the coverslips were
189
incubated with lectin HPA from Helix pomatia 647 (1:100) diluted in 0.05% Triton X-100
190
with 1% BSA in PBS for 1 h after the secondary antibody labeling, followed by washing three
191
times with PBS before DAPI staining and mounting.
192
The Leica TCS SP8 confocal microscope, with four laser beams (405 nm, 488 nm, 552
193
nm, and 638 nm), was used for image acquisition. Two sequential scans were performed at
194
400 Hz, first using the 405 nm laser (bandpass from 405–450 nm) and the 638 nm laser
195
(collection from > 640 nm), followed by a scan using the 488 nm laser (bandpass from 488–
196
550 nm). A control slide that was not stained with the primary antibody was used to set the
197
gain and offset values for each cell/antibody group.
198
The colocalization assessment between the Golgi staining and DS-epi1 was performed
199
in Huygens Professional 17.10.0 (Scientific Volume Imaging, Hilversum, The Netherlands).
200
Each of the five fields for the tested breast cancer cell lines had a region of interest (ROI)
11 201
generated in the red channel (i.e., the Golgi staining), with an established threshold. The
202
Costes method was applied for the background threshold prior to calculation of the Manders’
203
Overlap Coefficient (MOC), in which higher colocalization between DS-epi1 and the Golgi
204
staining is observed when MOC is nearest 1.
205
2.8 Data analysis and statistical treatment
206
Data were analyzed using GraphPad Prism 8.0 (San Diego, California, USA) and
207
Microsoft Office Excel 2016 (Redmond, Washington, USA). Different statistical tests were
208
applied, including one- and two-way analysis of variance (ANOVA), unpaired Student’s t-
209
test, and multiple Student’s t-tests. Statistically significant comparisons were set to the 5%
210
level, and data were plotted as the mean ± standard error of the mean (SEM).
211
3 RESULTS
212
3.1. SKBR3 cells grow more rapidly than MCF7 or MDA-MB-231
213
The initial aim of this study was to analyze and compare the expression of DS-epi1
214
among three different breast tumor cell lines, MCF7, MDA-MB-231 and SKBR3. However,
215
during routine cell culture, SKBR3 cells clearly demonstrated different growth behavior
216
compared with the other cell lines, which was indicated by the cell MTT assay. This assay
217
was performed using MDA-MB-231, MCF7, and SKBR3 cells at 24-, 48-, 72-, and 96-h time
218
points, as shown in Figure 1. The results clearly indicated that the SKBR3 cell line showed
219
increased OD570, with an exponential growth phase after 48 h, when compared with the other
220
tested cell lines. MDA-MB-231 and MCF7 cells showed similar growth curves, with a slow
221
growth curve that entered a saturation phase after 72 h, in contrast with SKBR3 cells. This
222
experiment was unexpected because MDA-MB-231 cells are classified as triple-negative (ER-
223
/PR-/HER2-) cells, which are commonly associated with increased genetic instability and
224
aggressiveness and a worse prognosis [34].
12 225
3.2. One variant of SKBR3 cells shows an entirely different growth curve and
226
expresses a different pattern of GAGs
227
These results led us to suspect that the SKBR3 cells used in this study (Figure 1) could
228
be modified (SKBR3m), as it was used at high passage levels (> 50). In an attempt to further
229
investigate this characteristic, a recently acquired ATCC vial (SKBR3a) was used, and the
230
cells were compared. The MTT assay was employed to assay the growth behavior of both
231
variants, using three time points, 24, 48 and 72 h (Figure 2). SKBR3m cells displayed erratic
232
growth, showing an exponential pattern, in contrast with the linear growth observed for
233
SKBR3a cells. This result suggested that SKBR3m may represent an aggressive variant of
234
SKBR3 cells.
235
We then investigated the GAG distributions in both SKBR3 cells, as shown in Figure
236
3. Apparent differences in CS and DS distributions were observed between SKBR3a and
237
SKBR3m cells. Although the CS content was low in SKBR3m cells, the DS content was high,
238
which was the opposite pattern of that observed for SKBR3a cells, where CS was expressed at
239
high levels, whereas DS was difficult to detect.
240
Therefore, we hypothesized that the different CS/DS contents might be associated with
241
the apparent difference in the growth curves between SKBR3m and SKBR3a cells, and we
242
proposed to investigate the role played by DS-epi1 in SKBR3m cells. All further
243
investigations utilized SKBR3m cells in all biological assays used to compare these cells with
244
other breast cancer cell lines.
245
3.3 Dermatan Sulfate epimerase 1 is expressed at higher levels in SKBR3m than
246
in other tested cell lines.
247
The expression level of DS-epi1 was investigated in MCF7 (luminal), MDA-MB-231
248
(triple-negative), and SKBR3m (HER2-positive) breast cancer cell lines. qPCR was used to
13 249
examine the expression levels of DS-epi1, the principal DS PGs, decorin, and biglycan
250
(Figure 4), relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression
251
levels. SKBR3m cells expressed significantly higher levels of DS-epi1 (p < 0.0001) than
252
either MDA-MB-231 (p = 0.003) or MCF7 (p = 0.0002) cells, according to Dunnett’s test. A
253
multiple Student’s t-test approach revealed that MCF7 cells expressed less DS-epi1 and
254
decorin than MDA-MB-231 (p < 0.0001) and SKBR3m (p = 0.0204) cells, indicating a
255
relationship between this PG and epiDS expression. Heparan sulfate epimerase (HS-epi)
256
expression was analyzed as a control and showed an independent pattern compared with
257
either DS-epi1 or decorin, as expected (Figure 4).
258
3.4. SKBR3m cells have lower protein levels of Dermatan Sulfate epimerase 1
259
than the other cell lines
260
The flow cytometry analysis was performed using specific antibodies to quantify the
261
protein levels of DS-epi1, HS-epi and decorin (Figure 5). Unexpectedly, MCF7 cells showed
262
higher protein expression levels of DS-epi1 than SKBR3m and MDA-MB-231 cells.
263
However, no differences in the protein expression levels of HS-epi were observed when
264
comparing the three breast cancer cell lines. MCF7 and SKBR3m cells displayed higher
265
expression levels of decorin than MDA-MB-231 cells.
266
The dot plot graph (Figure 6) allowed us to verify the existence of different cell
267
populations distributed among the studied cell lines, emphasizing a double-positive
268
population (decorin+/DS-epi1+). MCF7 cells contained the most double-positive cells (Q2 =
269
96.4%), followed by SKBR3m (Q2 = 88.8%) and MDA-MB-231 (Q2 = 86.1%) cells. The
270
existence of a decorin+/HS-epi+ population was invariable among the cell lines, with
271
approximately 90% of cells comprising this double-positive population.
14 272
3.5 Dermatan Sulfate epimerase 1 localizes to the Golgi apparatus in SKBR3m
273
cells but little in other cell lines
274
Immunofluorescence assays and confocal microscopy of MDA-MB-231, MCF7, and
275
SKBR3m cells showed the subcellular localizations of DS-epi1 and HS-epi proteins. Cells
276
were also labeled with DAPI and phalloidin-647 to identify the nucleus and the cytoskeleton,
277
respectively, as shown in Figure 7. Immunofluorescence quantification was analyzed by
278
ANOVA, with the Holm Šidák test, and the data revealed that SKBR3m cells presented the
279
lowest DS-epi1 protein contents among the three tested cell lines, whereas MCF7 cells had
280
the highest content (Figure 8), in contrast with the qPCR data.
281
The results also indicated a cytoplasmic localization for DS-epi1 in MDA-MB-231
282
and MCF7 cells, whereas in SKBR3m cells, the localization of this enzyme appears to be
283
perinuclear, with some vesicular stainings in the cytoplasm, suggesting Golgi apparatus
284
localization (Figure 7).
285
A more diffuse and cytoplasmic distribution for HS-epi was observed in MCF7 and
286
MDA-MB-231 cells (Figure 7), whereas localization to the Golgi apparatus was visualized in
287
SKBR3m cells, similar to the localization pattern observed for DS-epi1 (Figure 9).
288
An additional assay employing a Golgi apparatus-specific marker, the lectin HPA
289
from Helix Pomatia 647, was used to verify the specific localization of DS-epi1 in this
290
organelle, whose results can be viewed in Figure 9. This experiment revealed that MCF7 and
291
MDA-MB-231 cells express DS-epi1 primarily in the cytoplasm, whereas DS-epi1 is strictly
292
contained within the Golgi apparatus in SKBR3m cells, which is an organelle involved in the
293
post-translational processing of proteins (Figure 9A).
294
The colocalization analysis between the Golgi staining and DS-epi1 indicated that
295
SKBR3m cells had the highest degree of colocalization of this epimerase in the analyzed
15 296
organelle (0.863 ± 0.007), as shown in Figures 9B and 9C. Although MCF7 and MDA-MB-
297
231 displayed a certain degree of colocalization between both channels, it was 34.8% and
298
18.1% less intense, respectively, in comparison to SKBR3m.
299
3.6 SKBR3m cells, but not MCF7 and MDA-MB-231 cells, synthesize DS
300
After evaluating that DS-epi1 was mainly in the Golgi Apparatus of SKBR3m cells,
301
while in MCF7 and MDA-MB-231, the enzyme was mislocalized to the cytoplasm, it would
302
be worth to investigate the DS synthesis in these cells. To this end, metabolic labeling with
303
Na235SO4 was performed in these cell lines, and the total 35S-GAGs obtained. The GAGs
304
were then incubated with the specific chondroitinase AC and chondroitinase ABC.
305
Chondroitinase AC hydrolyzes β-(1-4) glycosidic bonds between N-acetyl-hexosamine and
306
glucuronic acid. Therefore, it is capable of hydrolyzing chondroitin-4-sulfate, chondroitin-6-
307
sulfate and hyaluronic acid to its unsaturated disaccharides-4-sulfated (∆U-GalNAc4S), -6-
308
sulfated (∆U-GalNAc6S) and non-sulfated (∆U-GlcNAc), respectively. Chondroitinase ABC
309
hydrolyzes β-(1-4) glycosidic bonds between N-acetyl-hexosamine and GlcA or IdoA acid. It
310
hydrolyzes chondroitin-4-sulfate, chondroitin-6-sulfate (∆U-GalNAc4S and ∆U-GalNAc6S),
311
DS (∆U-GalNAc4S) and hyaluronic acid (∆U-GlcNAc). After incubation with these enzymes,
312
the products were subjected to agarose gel electrophoresis, as shown in Figure 10.
313
Agarose gel electrophoresis can retain only larger polymers, not the digested products
314
by the enzymes. Disaccharides are unable to be identified in this experiment. Therefore, DS is
315
hydrolyzed by chondroitinase ABC but is resistant to chondroitinase AC so that this specific
316
GAG can be visualized in this gel. HS is resistant to both chondroitinases AC and ABC, so it
317
is detected in its intact form in this gel, as can be seen in Figure 10. The agarose gel
318
electrophoresis was able to identify a DS band only in SKBR3m cells, which does not occur
319
in MCF7 or MDA-MB-231 cells.
16 320
4 DISCUSSION
321
Breast cancer is a disease that currently affects a large number of people, primarily
322
females, and can be highly lethal, affecting individuals of all age groups [1, 35]. DS-epi1 is
323
the limiting enzyme for DS biosynthesis and acts on the GluA residues in CS chains in the
324
Golgi apparatus, converting them to IdoA [24, 26]. DS is a GAG that plays essential roles in
325
the modulation of oncogenic mechanisms, such as acting as a co-receptor for various growth
326
factors, including hepatocyte growth factor (HGF), fibroblast growth factor type 2 (FGF-2),
327
and platelet-derived growth factor (PDGF), all of which are crucial for the progression of
328
breast tumors. Its function as co-receptor is due to the existence of IdoA residues, which
329
confer greater flexibility to the molecule and favor increased binding specificity to these
330
growth factors [36].
331
We investigated the properties of DS-epi1 in three breast cancer cell lines: MCF7
332
(luminal A, ER+/PR+), MDA-MB-231 (triple-negative) and SKBR3 (HER2+). DS has been
333
shown to play a crucial aspect both in MCF7 and MDA-MB-231. In MDA-MB-231, the cells'
334
ability to synthesize full chains of CS/DS would affect these cells’ viability, motility, and
335
adhesion to matrix substrates [37]. Regarding MCF7, the ability of these cells to interact with
336
DSPGs, such as decorin or biglycan, would influence its viability and how the cells would
337
interact with growth factors, such as the vascular endothelial growth factor-165 (VEGF165)
338
[36]. We, therefore, selected both these cell lines to research the aspects of DS-epi1 but felt
339
the need to add a HER2+ cell line, as HER2 positivity represents 15–20% of breast cancers
340
[38]. SKBR3 was selected as it is one of the most widely used HER2+ cell lines [39].
341
We analyzed the localization of DS-epi1 in these breast cancer cells and assessed its
342
product, DS. We verified that the SKBR3m breast cancer cell line showed higher gene
343
expression levels of DS-epi1 than the other breast cancer cell lines (MCF7 and MDA-MB-
17 344
231). DS-epi1 has been found to be overexpressed in glioblastomas, as assessed by tissue
345
immunohistochemistry [40]. The same study further demonstrated that DS-epi1
346
overexpression was associated with advanced tumor grade and poor survival. DS-epi1
347
knockdown in glioblastoma cells demonstrated that this enzyme was responsible for
348
suppressing cell proliferation, migration, and invasion. In contrast, the induction of DS-epi1
349
overexpression increased malignant phenotypes and tumoral growth in vivo [40]. DS-epi1 is
350
also highly expressed in esophageal squamous cell carcinoma cells, which exhibit a four to
351
five-fold overexpression in enzyme activity compared with healthy tissue. In this tumor type,
352
DS-epi1 was localized both in the stroma surrounding the tumor and in the tumor cells, and
353
high DS-epi1 expression was also shown to be correlated with increased DS production [41].
354
Other studies have demonstrated that the overexpression of DS in esophageal squamous cell
355
carcinoma cells acts synergistically with HGF by stimulating tumoral migration and invasion.
356
This effect may be the result of the co-receptor abilities of DS with HGF and its respective
357
cellular receptor (c-Met), potentiating the growth factor’s effects [42].
358
Curiously, in our work, MCF7 cells presented higher DS-epi1 protein expression
359
levels than the other cell lines, although this effect was expected for the SKBR3m cell line
360
due to the higher gene expression of this enzyme in this specific cell line. The MCF7 and
361
SKBR3m cell lines both showed significantly increased expression levels of the decorin PG
362
core protein, as detected by flow cytometry.
363
We also examined DS-epi1 localization in SKBR3m, MCF7, and MDA-MB-231 cell
364
lines, using immunofluorescence assays with specific antibodies and confocal microscopy.
365
The SKBR3m cell line was the only cell line to show DS-epi1 content in the perinuclear
366
compartment, suggesting a location to the Golgi apparatus. This localization was further
367
confirmed by the immunofluorescence co-localization assay between the DS-epi1 enzyme and
368
Lectin-HPA (Helix pomatia agglutinin), a specific cellular marker for the Golgi apparatus.
18 369
The MCF7 and MDA-MB-231 cell lines showed cytoplasmic localization of the enzyme,
370
which could explain the increased detection of DS-epi1 by flow cytometry and quantitative
371
immunofluorescence.
372
From the literature, we know that the biosynthesis of GAGs, such as CS, occurs in the
373
endoplasmic reticulum and Golgi apparatus, starting with the addition of a tetrasaccharide and
374
followed by the consecutive addition of hexosamine and a non-nitrogen sugar. For the
375
formation of CS, these sugars correspond with N-acetylgalactosamine and glucuronic acid,
376
respectively. The formation of DS chains involves the conversion of GluA in CS chains to
377
IdoA, through the activity of the DS-epi1 enzyme [21-23]. Active DS-epi1 requires an
378
optimal pH of 5.5 and the presence of divalent cations, especially Mn²⁺ and the weakly
379
activating Ca²⁺ and Mg²⁺ cations. These conditions can be found within the Golgi apparatus,
380
which favors maximal enzyme activity in this organelle [43]. Nevertheless, previous studies
381
have observed that a mere change of 0.2–0.4 pH units in the Golgi luminal pH may promote
382
the mislocalization of several Golgi enzymes, such as α(2,3)-sialyltransferase, to endosomal
383
compartments [44]. Other glycosylation enzymes have also faced similar mislocalization to
384
endosomes or the plasma membrane in similar conditions [45, 46]. MCF7 has been shown to
385
be unable to acidify the Golgi lumen, leading to its alkalinization and mislocalization of
386
several of this organelle’s resident enzymes; a context also investigated in selected colorectal
387
cell lines (e.g., HT-29, SW-48, T84 and Caco-2) [47, 48]. Although there have been no
388
researches regarding how DS-epi1 localization may be affected by alkalinization of the Golgi
389
Apparatus, it is of our belief that certain cancer cell lines which may face it, may lose the
390
epimerization process related to DS synthesis, as suggested here.
391
Among the cell lines examined, SKBR3m was the only cell line containing DS after 35
392
the metabolic labeling of
S-GAGs and characterization with chondroitinase enzymes. As
393
mentioned earlier, DS is implicated in various cancer-promoting processes, such as cell
19 394
proliferation and metastasis. Our data showed increased SKBR3m cell growth, which differed
395
from the other cell lines analyzed, leading us to believe that this phenomenon could be
396
associated with the presence of DS.
397
Our results showed higher gene expression levels for DS-epi1 in SKBR3m cells and
398
the specific subcellular localization of the enzyme to the Golgi apparatus and the presence of
399
35
400
pattern. Our results showed that the localization of DS-epi1 in the Golgi apparatus is of great
401
importance for DS synthesis in breast cancer cell lines, suggesting that in cells where this
402
localization was not observed (e.g., cytoplasmic localization), the formation of DS chains
403
would be compromised. Thus, we suggest that the synthesis of DS could be associated with
404
breast cancer cell growth. The investigation of SKBR3 cells in the present study, which
405
resulted in the identification of the modified cell line, was fundamental to these results. The
406
changes in increased growth, combined with the enhanced synthesis of DS, a compound that
407
has been directly associated with oncogenicity, presents the opportunity to study interactions
408
between DS and critical proteins in the tumor microenvironment (e.g., growth factors,
409
chemokines, and cytokines), and we believe that DS deserves better characterization. The
410
importance of this study is to present a suitable model for breast cancer tumorigenesis and to
411
investigate new therapeutic targets that could be related to the “switch” in DS synthesis
412
observed in this study.
413
CONCLUSION
S-DS in these cells, which also demonstrated a more pronounced cellular proliferation
414
Our results showed that, in SKBR3m cells, the Golgi localization of DS-epi1 was a
415
determinant for DS synthesis. In the MCF7 and MDA-MB-231 cell lines, the cytoplasmic
416
localization of DS-epi1 may be associated with the lack of DS synthesis. However, the
417
decorin PG core protein was still synthesized, especially in SKBR3m and MCF7 cells.
20 418
Moreover, SKBR3m cells were the most viable cell line, and the presence of DS in this cell
419
line may contribute to the proliferative aspects in this particular in vitro model.
420
CONFLICT OF INTEREST
421
422
The authors report no conflict of interest in the content presented in this article.
ACKNOWLEDGMENTS
423
The authors acknowledge Caroline Z. Romera and Elizabeth N. Kanashiro
424
(INFAR/UNIFESP, Brazil) for their confocal technical assistance.
425
FUNDING
426
This work was supported by Fundação de Amparo à Pesquisa do Estado de São
427
Paulo (FAPESP, grant number 2016/18066-6 to LT), Conselho Nacional de Desenvolvimento
428
Científico e Tecnológico (CNPq, fellowship to EGX) and, in part, by the Coordenação de
429
Aperfeiçoamento de Pessoal de Nível Superior — Brasil (CAPES) — Finance Code 001,
430
including a fellowship to EL.
431
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25 520
FIGURE CAPTIONS
521
Figure 1. SKBR3m cells grow faster than MDA-MB-231 and MCF7 cells. The MTT assay
522
was performed at 24, 48, 72, and 96 h. The sample size was n = 16. Data were analyzed by
523
two-way ANOVA with Dunnett’s test. In this particular graph, data is displayed as mean ±
524
standard deviation (SD). * p < 0.05 vs. MDA-MB-231 and MCF7; **** p < 0.0001 vs. MDA-
525
MB-231 and MCF7.
526
Figure 2. The variant SKBR3m grows faster than SKBR3a. The MTT assay was
527
performed at 24, 48, and 72 h. For each time point, an unpaired Student’s t-test was applied to
528
compare OD570 scores between variants. The sample size was n = 24. In this particular graph,
529
data is displayed as mean ± SD. A linear fit was obtained for the variant SKBR3a (R2 =
530
0.928), and an exponential curve was fitted for SKBR3m (R2 = 0.934). * p < 0.05;**** p <
531
0.0001 vs. SKBR3 variants.
532
Figure 3. SKBR3m, and not SKBR3a, synthesizes DS and displays a GAG profile in the
533
cellular medium. (A) For each SKBR3 variant, the
534
extract (C) or medium (M), and after electrophoresis, the CS, DS, and HS contents were
535
analyzed. (B) Each GAG content was quantified and normalized against the total protein
536
quantity in the cell. No GAGs were detected in the medium from SKBR3a cells.
537
Figure 4. SKBR3m cells have a higher gene expression of DS-epi1 and decorin, in
538
comparison to MCF7 and MDA-MB-231 cells. All genes were assessed by RT-qPCR, in
539
which the sample size was n = 5–6. Data were analyzed by two-way ANOVA, with Dunnett’s
540
test. **** p < 0.0001 SKBR3m vs. MCF7; #### p < 0.0001 SBR3 vs. MDA-MB-231; and
541
$$$$ p < 0.0001 MCF7 vs. MDA-MB-231.
542
Figure 5. Concerning the protein expression, MCF7 displays higher DS-epi1 expression,
543
although both MCF7 and SKBR3m cells have high expression of decorin. The flow
35
S-GAGs were assessed in the cell
26 544
cytometry assay was used to analyze the expression profiles of DS-epi1, HS-epi, and decorin
545
proteoglycan. The sample size was n = 4–8. (A) Data were analyzed by two-way ANOVA,
546
with the Holm Šidák test. * p < 0.05; **** p < 0.0001 vs. MDA-MB-231. (B) The histograms
547
show cell population distributions in the flow cytometry experiment, in which unfilled
548
histograms are negative controls from unstained samples.
549
Figure 6. MCF7 and SKBR3m have a higher population of cells that both express DS-
550
epi1 and decorin. The flow cytometry assay analyzed DS-epi1, HS-epi, and decorin
551
proteoglycan expression. Decorin proteoglycan was stained with Alexa 633 and is shown in
552
the y-axis, and either DS-epi1 or HS-epi was labeled with Alexa 488 and is displayed on the
553
x-axis. In red, a representative experimental sample is shown for each cell line; and in blue, a
554
negative control.
555
Figure 7. SKBR3m has a more perinuclear localization of DS-epi1, as opposed to the
556
cytoplasmic display in MDA-MB-231 and MCF7. The immunofluorescence assay was
557
performed in MDA-MB-231 (a and d), MCF7 (b and e) and SKBR3m (c and f) cells
558
assessing DS-epi-1 (a, b or c) or HS-epi (d, e, or f) protein content. Cells were stained with
559
DAPI (1) or F-actin (3), in addition to DS-epi1 or HS-epi (2) labeling. The composite image
560
(4) is also exhibited. The scale bar represents 200 µm, and 7 to 9 fields were analyzed.
561
Figure 8. Although SKBR3m has a more precise localization of DS-epi1, MCF7 displays
562
a higher quantity of it. The data from the immunofluorescence assay was quantified for DS-
563
epi1 and HS-epi content. The sample size was n = 7–9. Data were analyzed by two-way
564
ANOVA, with the Holm Šidák test. ** p < 0.01 vs. MDA-MB-231; ### p < 0.001 vs. MCF7.
565
Figure 9. SKBR3m cells have a higher Golgi colocalization of DS-epi1 than MCF7 and
566
MDA-MB-231. (A) Cells were imaged for DS-epi1 (2) and a Golgi apparatus cellular marker,
567
(lectin HPA, 3). The cells were also stained with DAPI (1). All channels were merged (4), and
27 568
the scale bar represents 50 µm. (B) The representatives' histograms display the colocalization
569
between pixels from the red channel (lectin HPA) and the green channel (DS-epi1) for each
570
tested cell line. (C) The Manders’ Overlap Coefficient (MOC) indicates the degree of
571
colocalization between DS-epi1 with the lectin HPA (i.e., the Golgi marker) for each tested
572
cell line. Data were analyzed with multiple Student’s t-tests. * p < 0.05, **** p < 0.0001 vs.
573
SKBR3m.
574
Figure. 10. Only SKBR3m synthesize DS when compared to MDA-MB-231 and MCF7
575
cells. The assay was performed by labeling GAGs with 35S, purifying them with ethanol, and
576
proteolytically degrading the PG core protein with alcalase. The products were then digested
577
with chondroitinase AC or ABC and separated by electrophoretic mobility in agarose gels in
578
PDA buffer. Only SKBR3m cells displayed DS content, differing from the other cell lines that
579
only contained HS and CS (not shown).
HIGHLIGHTS • • • •
A SKBR3 cell line from a high passage (SKBR3m) is highly proliferative in comparison to MCF7 and MDA-MB-231 cells; The SKBR3m cells were the only to synthesize dermatan sulfate; Dermatan sulfate epimerase 1 (DS-epi1) was found in the Golgi Apparatus only in SKBR3m cells; MCF7 and MDA-MB-231 cells presented DS-epi1 mislocalized.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
S0008-6215(19)30581-6
DOI:
https://doi.org/10.1016/j.carres.2020.107906
Reference:
CAR 107906
To appear in:
Carbohydrate Research
Received Date: 3 October 2019 Revised Date:
10 December 2019
Accepted Date: 3 January 2020
Please cite this article as: E. Listik, Everton.Galvã. Xavier, M.A.d. Silva Pinhal, L. Toma, Dermatan sulfate epimerase 1 expression and mislocalization may interfere with dermatan sulfate synthesis and breast cancer cell growth, Carbohydrate Research (2020), doi: https://doi.org/10.1016/ j.carres.2020.107906. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
1
Dermatan sulfate epimerase 1 expression and mislocalization may interfere with dermatan sulfate synthesis and breast cancer cell growth Eduardo Listik#, Everton Galvão Xavier#, Maria Aparecida da Silva Pinhal & Leny Toma*
Affiliation: Department of Biochemistry, Universidade Federal de São Paulo, Rua Três de Maio, 100, São Paulo, SP, Brazil. CEP: 04044-020. [email protected]; [email protected]; [email protected]; [email protected].
Corresponding author: Leny Toma, Ph.D. Department of Biochemistry Universidade Federal de São Paulo Rua Três de Maio, 100, 5th floor, CEP: 04044-020 São Paulo, SP, Brazil. Phone: +55-11-5573-6407 Email: [email protected]
#
Authors share equal contribution.
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ABSTRACT
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Dermatan sulfate (DS) is a glycosaminoglycan (GAG) that is produced through the
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epimerization of the glucuronic acid on chondroitin sulfate into iduronic acid (IduA) by
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dermatan sulfate epimerase (DS-epi) 1 and 2. Proteoglycans (PGs) play essential physiological
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and pathological roles during cellular development, proliferation, differentiation, and cancer
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metastasis. DS proteoglycans play vital roles during the process of tumorigenesis, due to the
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increased flexibility of the polysaccharide chain in the presence of IduA residues, which
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facilitate specific interactions with proteins, such as growth factors, cytokines, and angiogenic
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factors. Furthermore, DS-epi is highly expressed in many tumors, especially in esophageal
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squamous cell carcinoma. This study aimed to investigate the expression of DS-epi1 in multiple
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breast cancer cell lines, including MCF7 (luminal A), MDA-MB-231 (triple-negative) and
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SKBR3 (human epidermal growth factor receptor 2-positive), and its involvement in cancer
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progression. A SKBR3 variant, SKBR3m, presented the most erratic cell growth pattern when
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compared with those for MCF7 and MDA-MB-231. Moreover, SKBR3m cells demonstrated
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the highest level of DS-epi1 gene expression and higher
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protein level, MCF7 cells displayed the highest protein level for DS-epi1, whereas MDA-MB-
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231 cells had the lowest level. DS-epi1 was found in vesicles and in the perinuclear
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compartment only in SKBR3m cells, suggesting localization in the Golgi apparatus in these
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cells, in contrast with the cytoplasmic localization observed in MCF7 and MDA-MB-231 cells.
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The cytoplasm location of DS-epi1 likely compromised the formation of DS chains, but the
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core protein was detected using a decorin antibody. Golgi-specific labeling confirmed the
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localization of DS-epi1 in SKBR3m cells at the Golgi apparatus, indicating that the location of
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the enzyme was a determinant for the synthesis of DS in this cell line, suggesting that DS may
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play a decisive role in the tumor growth observed in this breast cancer cell line.
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S-DS content. However, at the
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Keywords: Breast cancer; cancer biology; dermatan sulfate; decorin proteoglycan;
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Golgi apparatus.
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1 INTRODUCTION
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Breast cancer is one of the most prevalent types of tumors among women worldwide,
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with more than 1.7 million new registered cases and 522,000 deaths every year. The incidence
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of breast cancer has continued to grow, and more than 3.2 million new cases are expected by
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2050 [1]. Breast cancer pathology can be classified according to morphologic, histologic, and
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molecular parameters. Morphologically, breast tumors can be categorized as infiltrating ductal
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carcinomas of no particular type, infiltrating lobular and tubular carcinomas, mucinous
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carcinomas, and many others. Histological classifications allow the sub-categorization of
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breast tumors, using characteristics such as cellular differentiation, nuclear pleomorphism,
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and mitotic counts. At the molecular level, breast tumors can be classified according to the
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presence or absence of estrogen receptor (ER), progesterone receptor (PR), or human
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epidermal growth factor receptor type 2 (HER2). The majority of breast tumors are ER-
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positive; however, many subgroups can be characterized, such as luminal A and B tumors,
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which are ER+/PR+ [2-4].
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Proteoglycans (PGs) are macromolecules that possess a protein core, to which one or
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more glycosaminoglycan (GAG) chains are covalently attached [5]. Such molecules have
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essential biological functions during vascularization and cancer metastasis and assist many
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signaling proteins in the extracellular matrix [6, 7]. During tumoral development and growth,
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the expression of PGs is markedly modified, frequently upregulated, and many PGs influence
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or inhibit cell proliferation, depending on the tissue type [8-10].
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Chondroitin sulfate (CS) is a GAG that is naturally found in the extracellular matrix of
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connective tissue, either on the cell surface or in intracellular compartments. A disaccharidic
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unit, composed of D-glucuronic acid (GlcA, β-1,4) and N-acetyl-D-galactosamine (GalNAc,
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β-1,3), can be found in CS [11, 12]. Dermatan sulfate (DS), in contrast, possesses the GlcA
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epimer iduronic acid (IdoA). Both GAGs are structurally similar, and they appear
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concomitantly in PGs [7, 13].
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GAG biosynthesis begins at the rough endoplasmic reticulum (RER) and proceeds to
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the Golgi apparatus. In the RER, GAG synthesis begins with the formation of a
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tetrasaccharidic sequence of GlcA, β1→3, D-galactose (Gal), β1→3, Gal, β1→4, xylose (Xyl),
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β1→ [14-16]. After the addition of xylose and D-galactose, the synthetic process is transferred
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to the Golgi apparatus [17, 18]. The fifth added sugar determines whether the GAG will
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become a CS/DS or heparan sulfate (HS)/heparin (Hep) [19, 20]. The extension of CS occurs
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through the activity of the chondroitin synthase enzyme. CS chains in mammals are
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commonly sulfated on C-4 or C-6 positions of GalNAc, generating the isomers chondroitin-4-
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sulfate or chondroitin-6-sulfate, respectively [21-23]. GlcA/IdoA can also be sulfated on the
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C-2 position, either in CS or DS [24, 25]. Epimerization proceeds sulfation, during which
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dermatan epimerase (DS-epi) 1 and 2 act on the C-5 of GlcA to generate an IdoA moiety [26,
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27].
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PGs play significant roles in several physiological and pathological cellular processes.
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The results obtained in the present study may indicate that higher expression levels of DS-
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epi1 in cells derived from breast tumors could be linked with higher growth rates. However,
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the cells that express higher levels of DS-epi1 do not show higher DS-epi1 protein levels;
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instead, DS-epi1 presents with a more specific localization in vesicles. This result led us to
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question whether the vesicle-localized DS-epi1 found in cells with higher levels of DS-epi1
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expression is functional.
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2 MATERIALS AND METHODS
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2.1 Materials
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Anti-HS-epi (polyclonal rabbit, IgG, NBP2-31872) and anti-DS-epi1 (polyclonal
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rabbit, IgG, NBP1-87962) antibodies were purchased from Novus Biologicals (Centennial,
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Colorado, USA). Anti-decorin (monoclonal mouse, IgG, MA5-29240) was purchased from
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Thermo Fisher (Waltham, Massachusetts, USA). All secondary antibodies, including anti-
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rabbit Alexa Fluor 488 (cat. A-21206), anti-mouse Alexa Fluor 633 (cat. A-21050),
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phalloidin-647 (cat. A22287), and lectin HPA from Helix Pomatia-647 (cat. L32454), were
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purchased from Thermo Fisher. RPMI1640, Dulbecco’s Modified Eagle Medium (DMEM),
81
and fetal bovine serum (FBS) were all obtained from Gibco® (from Thermo Fisher).
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2.2 Cell culture
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MDA-MB-231 (ATCC® HTB-26TM), MCF7 (ATCC® HTB-22TM) and SKBR3
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(ATCC® HTB-30TM) cells were purchased from the American Type Culture Collection
85
(ATCC, Manassas, Virgínia, USA). MDA-MB-231 and MCF7 cells were cultivated in
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DMEM, whereas SKBR3 cells were cultivated in RPMI1640. SKBR3 cells were used in
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specific assays: SKBR3m from a high passage (> 50), and SKBR3a from low passage levels
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(< 5), as it has been previously described that breast cancer cell lines may acquire a more
89
aggressive profile in higher passages [28-31]. All media were supplemented with 10% FBS
90
(v/v) and 1% penicillin/streptomycin (v/v) The cells were grown at 37 °C, in a humidified
91
atmosphere containing 5% CO2.
92
2.3 MTT assay
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The MTT assay was performed using the tetrazolium salt (MTT) reagent, as
94
previously described [32]. In brief, MDA-MB-231, MCF7, and both types of SKBR3 cells
95
were plated at a 3×103 cell/mL density in a 96-well plate. A 5 mg/mL sterile solution of MTT
96
in phosphate-buffered saline (PBS) was prepared, and 10 µL was added 4 h prior to cell
6 97
incubation for 24, 48, and 72 h. In some assays, a 96-h time point was also analyzed. At each
98
time point, the supernatant was removed from each well, and the formazan precipitate was
99
solubilized with 120 µL dimethylsulfoxide (DMSO). The plate was then briefly agitated, and
100
the absorbance of each well was read using a plate reader at 570 nm. The optical density of
101
blank wells was subtracted from each experimental well, and the results were normalized
102
against those for control wells to which MTT was not added.
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2.4
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electrophoresis
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S-Sulfate metabolic radiolabeling of GAGs and analysis by agarose gel
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MDA-MB-231, MCF7 and both types of SKBR3 cells were cultivated in either 35-
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mm dishes or 24-well plates until they reached 60% confluence. Then, they were incubated
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with Na235SO4 (100 µCi/mL) for 24 h. The medium was replaced with OptiMEM during the
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radioactive pulse.
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The medium was collected, and cells were washed twice with 2 mL cold PBS and
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collected by scraping in 500 µL 3.5 M urea buffer (pH 7.4). PGs in both the collected media
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and the cell extracts were proteolyzed with proteinase K at a final concentration of 100
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µg/mL, for 3 h at 40 °C, before being inactivated at 95 °C for 10 min. Alternatively, the PGs
113
from the cell extracts and media were precipitated with three volumes of ethanol, in the
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presence of 200 µg CS as a carrier, and incubated overnight at -20 °C. The solution was
115
centrifugated at 3,000 rpm for 10 min, and the precipitate was left to dry at a vacuum
116
concentrator. A proteolytic solution, consisting of 0.9 mM alcalase in 100 mM Tris-HCl (pH
117
7.4), was added and incubated for two days at 54 °C, followed by inactivation by boiling for 2
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h.
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In some experiments, after proteolysis, GAGs were digested with either chondroitinase
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AC (0.1 units in 20 mM Tris-HCl, pH 7.2, 2% glycerol), chondroitinase ABC (0.1 units in 50
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mM Tris-HCl, 60 mM sodium acetate, 0.02% of BSA (v/v)), both from Sigma Aldrich (San
122
Luis, Missouri, USA) or water, as a control. All samples were incubated overnight at 37 °C
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(for chondroitinases). Then, agarose electrophoresis was performed, as previously described
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[33]. In brief, 5 µL of each sample were applied to agarose gel slabs in 0.05 M 1,3-
125
diaminepropane acetate, pH 9.0. Afterward, GAGs in the gel slabs were precipitated for 1 h
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with 0.2% cetyltrimethylammonium bromide (v/v), and then air-dried and stained with a
127
solution composed of 0.1% toluidine blue, 50% ethanol, and 1% acetic acid. The gel was
128
destained in a solution composed of 50% ethanol and 1% acetic acid and exposed to a
129
phosphor screen for visualization and quantification using a Cyclone® PhosphorImager. The
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data, in counts per minute (cpm), were obtained by comparison with a standard radioactive
131
ink measured using a liquid scintillation counter (TRI-CARB 4910TR, Perkin-Elmer,
132
Waltham, Massachusetts, USA). The final value was normalized against total cell protein
133
content.
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2.5 RNA extraction and real-time quantitative reverse transcription PCR (RT-
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qPCR)
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MDA-MB-231, MCF7, and SKBR3m cells were cultivated in 60-mm dishes until 90%
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confluence was achieved. The medium was removed, and cells were incubated for 5 min with
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1 mL TRIzolTM reagent. The lysate solution was transferred to tubes and mixed with 500 µL
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chloroform by inversion. The tubes were centrifuged for 30 min, at 12,000 rpm, at 4 °C, and
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the aqueous phase was transferred to a new tube. RNA was precipitated with 500 µL
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isopropanol and sequentially washed with 1 mL 75% ethanol. The RNA pellet was left to dry
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in a vacuum concentrator and resuspended in 40 µL RNase-free water.
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Total RNA was quantified in a Nanodrop (NanoVueTM Plus, GE Healthcare, Chicago,
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Illinois, USA), and a sample containing 2 µg total RNA and 2 µM oligo(dT)15 primers in 5 µL
145
RNase-free water was incubated at 70 °C for 5 min and then stored at 4 °C to minimize the
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formation of RNA secondary structures. A 15 µL mixture, containing 667 µM dNTP mix, 4
147
mM MgCl2, 10.7 U/µL Improm-II reverse transcriptase, Improm-II reverse transcriptase
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buffer (1.33×) and 1.33 U/µL Ribolock RNAse Inhibitor (Thermo Fisher Scientific), was
149
added to each sample. The reverse transcription reaction conditions were as follows:
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annealing for 5 min at 25 °C, extension at 42 °C for 60 min, and enzyme inactivation at 70 °C
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for 10 min.
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cDNA was also quantified, and a quantitative PCR (qPCR) reaction mixture was
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prepared to contain 50 ng/mL cDNA, 500 nM forward primers, 500 nM reverse primers, and
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5 µL SYBR® Green PCR Master Mix (Applied Biosystems by Thermo Fisher), in a total
155
volume of 20 µL. The primers used in this assay are listed in Table 1. The qPCR conditions
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included an activation phase for 10 min at 95 °C, and 40 cycles of denaturation (15 s at
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95 °C), and annealing (1 min at 60 °C), with data collection occurring during the last step.
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Table 1. Primers used in RT-qPCR.
Gene
Forward and reverse primers (5’-NNN-3’)
F: 5’-TGTGTGCTGTATCCTGAGAACA-3’ Dermatan sulfate epimerase 1 R: 5’-CAAGGCGCATCTTTCACCAC-3’ F: 5’-TTCCAAAGTCTATGCACAGGAGC-3’ Heparan sulfate epimerase R: 5’-TCCACATTGTAGCCTTCAAAAGAC-3’ F: 5’-TGGACCGTTTCAACAGAGAGGCTT 3’ Decorin proteoglycan R: 5’-TGGACCACTCGAAGATGGCATTGA-3’ Biglycan proteoglycan
F: 5’-AAGCTCAACTACCTGCGCATCTCA-3’
9 R: 5’-ATCATCCTGATCTGGTTGTGGCCT-3’ F: 5’-TAGCACTGCCCTTGGAATTTGTGC-3’ Versican proteoglycan R: 5’-AGCGGAGACCAGTGTGAACTTGAT-3’ F: 5’-TCGACAGTCAGCCGCATCTTCTTT-3’ GAPDH R: 5’-ACCAAATCCGTTGACTCCGACCTT-3’
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2.6 Flow cytometry staining and data collection
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MDA-MB-231, MCF7, and SKBR3m cells were cultivated in 100-mm dishes until
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90% confluence was reached. The cells were detached using a 20-min incubation with 500
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µM ethylenediaminetetraacetic acid (EDTA). The cells were collected by centrifugation,
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distributed into tubes, and fixed with 200 µL 2% formaldehyde in PBS for 20 min, on ice.
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The cells were washed sequentially with PBS and 0.1 M glycine and then permeabilized with
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100 µL 0.01% saponin in PBS for 30 min on ice. After washing with PBS, the cells were
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incubated with 50 µL antibody solution at 1:100 dilution for 1 h 30 min (anti-DS-epi1, anti-
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HS-epi, or anti-decorin), diluted in 1% BSA in PBS. After washing with PBS, 50 µL
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secondary antibody solution was added, containing either anti-rabbit Alexa Fluor 488 (1:200)
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or anti-mouse Alexa Fluor 633 (1:200), for 40 min. The cells were resuspended in 200 µL
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PBS and counted with a BD FACSCalibur (BD Biosciences). The fluorescence geometric
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mean average of the cell population was used to compare flow cytometry data among cell
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lines, and histograms were plotted for visual assessments.
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2.7 Immunofluorescence staining and confocal imaging
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MDA-MB-231, MCF7, and SKBR3m cells were plated at a 2×105 cell/mL density on
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sterile borosilicate coverslips in a 24-well plate and incubated overnight. The supernatant was
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removed, and cells were washed with cold PBS, followed by fixation with 4%
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paraformaldehyde that was pre-warmed to 37 °C for 15 min. The coverslips were washed
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three times with PBS, one time with 0.1 M glycine, and once again with PBS, for 5 min each
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wash. The cells were then blocked and permeabilized with a solution containing 0.1% Triton
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X-100 and 3% BSA for 1 h. Anti-DS-epi1 (rabbit IgG, 1:100) or anti-HS-epi (rabbit IgG,
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1:150) were diluted in 0.05% Triton X-100 with 1% BSA in PBS were added to the cells and
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incubated overnight at 4 °C. The coverslips were washed three times with PBS for 10 min and
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incubated with anti-rabbit Alexa Fluor 488 (1:200) and phalloidin-647 (1:200) in 0.05%
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Triton X-100 with 1% BSA in PBS, for 1 h at room temperature. The cells were washed three
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times with PBS for 5 min and incubated with 4′,6-Diamidine-2′-phenylindole dihydrochloride
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(DAPI, 1:2,000 in PBS) for 15 min. The coverslips were washed five times with PBS for five
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minutes, dipped twice in cold type I water, mounted with FluoromountTM onto clean slides,
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and sealed with colorless nail polish. For Golgi apparatus staining, the coverslips were
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incubated with lectin HPA from Helix pomatia 647 (1:100) diluted in 0.05% Triton X-100
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with 1% BSA in PBS for 1 h after the secondary antibody labeling, followed by washing three
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times with PBS before DAPI staining and mounting.
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The Leica TCS SP8 confocal microscope, with four laser beams (405 nm, 488 nm, 552
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nm, and 638 nm), was used for image acquisition. Two sequential scans were performed at
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400 Hz, first using the 405 nm laser (bandpass from 405–450 nm) and the 638 nm laser
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(collection from > 640 nm), followed by a scan using the 488 nm laser (bandpass from 488–
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550 nm). A control slide that was not stained with the primary antibody was used to set the
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gain and offset values for each cell/antibody group.
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The colocalization assessment between the Golgi staining and DS-epi1 was performed
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in Huygens Professional 17.10.0 (Scientific Volume Imaging, Hilversum, The Netherlands).
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Each of the five fields for the tested breast cancer cell lines had a region of interest (ROI)
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generated in the red channel (i.e., the Golgi staining), with an established threshold. The
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Costes method was applied for the background threshold prior to calculation of the Manders’
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Overlap Coefficient (MOC), in which higher colocalization between DS-epi1 and the Golgi
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staining is observed when MOC is nearest 1.
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2.8 Data analysis and statistical treatment
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Data were analyzed using GraphPad Prism 8.0 (San Diego, California, USA) and
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Microsoft Office Excel 2016 (Redmond, Washington, USA). Different statistical tests were
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applied, including one- and two-way analysis of variance (ANOVA), unpaired Student’s t-
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test, and multiple Student’s t-tests. Statistically significant comparisons were set to the 5%
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level, and data were plotted as the mean ± standard error of the mean (SEM).
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3 RESULTS
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3.1. SKBR3 cells grow more rapidly than MCF7 or MDA-MB-231
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The initial aim of this study was to analyze and compare the expression of DS-epi1
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among three different breast tumor cell lines, MCF7, MDA-MB-231 and SKBR3. However,
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during routine cell culture, SKBR3 cells clearly demonstrated different growth behavior
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compared with the other cell lines, which was indicated by the cell MTT assay. This assay
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was performed using MDA-MB-231, MCF7, and SKBR3 cells at 24-, 48-, 72-, and 96-h time
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points, as shown in Figure 1. The results clearly indicated that the SKBR3 cell line showed
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increased OD570, with an exponential growth phase after 48 h, when compared with the other
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tested cell lines. MDA-MB-231 and MCF7 cells showed similar growth curves, with a slow
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growth curve that entered a saturation phase after 72 h, in contrast with SKBR3 cells. This
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experiment was unexpected because MDA-MB-231 cells are classified as triple-negative (ER-
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/PR-/HER2-) cells, which are commonly associated with increased genetic instability and
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aggressiveness and a worse prognosis [34].
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3.2. One variant of SKBR3 cells shows an entirely different growth curve and
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expresses a different pattern of GAGs
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These results led us to suspect that the SKBR3 cells used in this study (Figure 1) could
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be modified (SKBR3m), as it was used at high passage levels (> 50). In an attempt to further
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investigate this characteristic, a recently acquired ATCC vial (SKBR3a) was used, and the
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cells were compared. The MTT assay was employed to assay the growth behavior of both
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variants, using three time points, 24, 48 and 72 h (Figure 2). SKBR3m cells displayed erratic
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growth, showing an exponential pattern, in contrast with the linear growth observed for
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SKBR3a cells. This result suggested that SKBR3m may represent an aggressive variant of
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SKBR3 cells.
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We then investigated the GAG distributions in both SKBR3 cells, as shown in Figure
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3. Apparent differences in CS and DS distributions were observed between SKBR3a and
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SKBR3m cells. Although the CS content was low in SKBR3m cells, the DS content was high,
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which was the opposite pattern of that observed for SKBR3a cells, where CS was expressed at
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high levels, whereas DS was difficult to detect.
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Therefore, we hypothesized that the different CS/DS contents might be associated with
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the apparent difference in the growth curves between SKBR3m and SKBR3a cells, and we
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proposed to investigate the role played by DS-epi1 in SKBR3m cells. All further
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investigations utilized SKBR3m cells in all biological assays used to compare these cells with
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other breast cancer cell lines.
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3.3 Dermatan Sulfate epimerase 1 is expressed at higher levels in SKBR3m than
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in other tested cell lines.
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The expression level of DS-epi1 was investigated in MCF7 (luminal), MDA-MB-231
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(triple-negative), and SKBR3m (HER2-positive) breast cancer cell lines. qPCR was used to
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examine the expression levels of DS-epi1, the principal DS PGs, decorin, and biglycan
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(Figure 4), relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression
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levels. SKBR3m cells expressed significantly higher levels of DS-epi1 (p < 0.0001) than
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either MDA-MB-231 (p = 0.003) or MCF7 (p = 0.0002) cells, according to Dunnett’s test. A
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multiple Student’s t-test approach revealed that MCF7 cells expressed less DS-epi1 and
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decorin than MDA-MB-231 (p < 0.0001) and SKBR3m (p = 0.0204) cells, indicating a
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relationship between this PG and epiDS expression. Heparan sulfate epimerase (HS-epi)
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expression was analyzed as a control and showed an independent pattern compared with
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either DS-epi1 or decorin, as expected (Figure 4).
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3.4. SKBR3m cells have lower protein levels of Dermatan Sulfate epimerase 1
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than the other cell lines
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The flow cytometry analysis was performed using specific antibodies to quantify the
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protein levels of DS-epi1, HS-epi and decorin (Figure 5). Unexpectedly, MCF7 cells showed
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higher protein expression levels of DS-epi1 than SKBR3m and MDA-MB-231 cells.
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However, no differences in the protein expression levels of HS-epi were observed when
264
comparing the three breast cancer cell lines. MCF7 and SKBR3m cells displayed higher
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expression levels of decorin than MDA-MB-231 cells.
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The dot plot graph (Figure 6) allowed us to verify the existence of different cell
267
populations distributed among the studied cell lines, emphasizing a double-positive
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population (decorin+/DS-epi1+). MCF7 cells contained the most double-positive cells (Q2 =
269
96.4%), followed by SKBR3m (Q2 = 88.8%) and MDA-MB-231 (Q2 = 86.1%) cells. The
270
existence of a decorin+/HS-epi+ population was invariable among the cell lines, with
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approximately 90% of cells comprising this double-positive population.
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3.5 Dermatan Sulfate epimerase 1 localizes to the Golgi apparatus in SKBR3m
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cells but little in other cell lines
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Immunofluorescence assays and confocal microscopy of MDA-MB-231, MCF7, and
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SKBR3m cells showed the subcellular localizations of DS-epi1 and HS-epi proteins. Cells
276
were also labeled with DAPI and phalloidin-647 to identify the nucleus and the cytoskeleton,
277
respectively, as shown in Figure 7. Immunofluorescence quantification was analyzed by
278
ANOVA, with the Holm Šidák test, and the data revealed that SKBR3m cells presented the
279
lowest DS-epi1 protein contents among the three tested cell lines, whereas MCF7 cells had
280
the highest content (Figure 8), in contrast with the qPCR data.
281
The results also indicated a cytoplasmic localization for DS-epi1 in MDA-MB-231
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and MCF7 cells, whereas in SKBR3m cells, the localization of this enzyme appears to be
283
perinuclear, with some vesicular stainings in the cytoplasm, suggesting Golgi apparatus
284
localization (Figure 7).
285
A more diffuse and cytoplasmic distribution for HS-epi was observed in MCF7 and
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MDA-MB-231 cells (Figure 7), whereas localization to the Golgi apparatus was visualized in
287
SKBR3m cells, similar to the localization pattern observed for DS-epi1 (Figure 9).
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An additional assay employing a Golgi apparatus-specific marker, the lectin HPA
289
from Helix Pomatia 647, was used to verify the specific localization of DS-epi1 in this
290
organelle, whose results can be viewed in Figure 9. This experiment revealed that MCF7 and
291
MDA-MB-231 cells express DS-epi1 primarily in the cytoplasm, whereas DS-epi1 is strictly
292
contained within the Golgi apparatus in SKBR3m cells, which is an organelle involved in the
293
post-translational processing of proteins (Figure 9A).
294
The colocalization analysis between the Golgi staining and DS-epi1 indicated that
295
SKBR3m cells had the highest degree of colocalization of this epimerase in the analyzed
15 296
organelle (0.863 ± 0.007), as shown in Figures 9B and 9C. Although MCF7 and MDA-MB-
297
231 displayed a certain degree of colocalization between both channels, it was 34.8% and
298
18.1% less intense, respectively, in comparison to SKBR3m.
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3.6 SKBR3m cells, but not MCF7 and MDA-MB-231 cells, synthesize DS
300
After evaluating that DS-epi1 was mainly in the Golgi Apparatus of SKBR3m cells,
301
while in MCF7 and MDA-MB-231, the enzyme was mislocalized to the cytoplasm, it would
302
be worth to investigate the DS synthesis in these cells. To this end, metabolic labeling with
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Na235SO4 was performed in these cell lines, and the total 35S-GAGs obtained. The GAGs
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were then incubated with the specific chondroitinase AC and chondroitinase ABC.
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Chondroitinase AC hydrolyzes β-(1-4) glycosidic bonds between N-acetyl-hexosamine and
306
glucuronic acid. Therefore, it is capable of hydrolyzing chondroitin-4-sulfate, chondroitin-6-
307
sulfate and hyaluronic acid to its unsaturated disaccharides-4-sulfated (∆U-GalNAc4S), -6-
308
sulfated (∆U-GalNAc6S) and non-sulfated (∆U-GlcNAc), respectively. Chondroitinase ABC
309
hydrolyzes β-(1-4) glycosidic bonds between N-acetyl-hexosamine and GlcA or IdoA acid. It
310
hydrolyzes chondroitin-4-sulfate, chondroitin-6-sulfate (∆U-GalNAc4S and ∆U-GalNAc6S),
311
DS (∆U-GalNAc4S) and hyaluronic acid (∆U-GlcNAc). After incubation with these enzymes,
312
the products were subjected to agarose gel electrophoresis, as shown in Figure 10.
313
Agarose gel electrophoresis can retain only larger polymers, not the digested products
314
by the enzymes. Disaccharides are unable to be identified in this experiment. Therefore, DS is
315
hydrolyzed by chondroitinase ABC but is resistant to chondroitinase AC so that this specific
316
GAG can be visualized in this gel. HS is resistant to both chondroitinases AC and ABC, so it
317
is detected in its intact form in this gel, as can be seen in Figure 10. The agarose gel
318
electrophoresis was able to identify a DS band only in SKBR3m cells, which does not occur
319
in MCF7 or MDA-MB-231 cells.
16 320
4 DISCUSSION
321
Breast cancer is a disease that currently affects a large number of people, primarily
322
females, and can be highly lethal, affecting individuals of all age groups [1, 35]. DS-epi1 is
323
the limiting enzyme for DS biosynthesis and acts on the GluA residues in CS chains in the
324
Golgi apparatus, converting them to IdoA [24, 26]. DS is a GAG that plays essential roles in
325
the modulation of oncogenic mechanisms, such as acting as a co-receptor for various growth
326
factors, including hepatocyte growth factor (HGF), fibroblast growth factor type 2 (FGF-2),
327
and platelet-derived growth factor (PDGF), all of which are crucial for the progression of
328
breast tumors. Its function as co-receptor is due to the existence of IdoA residues, which
329
confer greater flexibility to the molecule and favor increased binding specificity to these
330
growth factors [36].
331
We investigated the properties of DS-epi1 in three breast cancer cell lines: MCF7
332
(luminal A, ER+/PR+), MDA-MB-231 (triple-negative) and SKBR3 (HER2+). DS has been
333
shown to play a crucial aspect both in MCF7 and MDA-MB-231. In MDA-MB-231, the cells'
334
ability to synthesize full chains of CS/DS would affect these cells’ viability, motility, and
335
adhesion to matrix substrates [37]. Regarding MCF7, the ability of these cells to interact with
336
DSPGs, such as decorin or biglycan, would influence its viability and how the cells would
337
interact with growth factors, such as the vascular endothelial growth factor-165 (VEGF165)
338
[36]. We, therefore, selected both these cell lines to research the aspects of DS-epi1 but felt
339
the need to add a HER2+ cell line, as HER2 positivity represents 15–20% of breast cancers
340
[38]. SKBR3 was selected as it is one of the most widely used HER2+ cell lines [39].
341
We analyzed the localization of DS-epi1 in these breast cancer cells and assessed its
342
product, DS. We verified that the SKBR3m breast cancer cell line showed higher gene
343
expression levels of DS-epi1 than the other breast cancer cell lines (MCF7 and MDA-MB-
17 344
231). DS-epi1 has been found to be overexpressed in glioblastomas, as assessed by tissue
345
immunohistochemistry [40]. The same study further demonstrated that DS-epi1
346
overexpression was associated with advanced tumor grade and poor survival. DS-epi1
347
knockdown in glioblastoma cells demonstrated that this enzyme was responsible for
348
suppressing cell proliferation, migration, and invasion. In contrast, the induction of DS-epi1
349
overexpression increased malignant phenotypes and tumoral growth in vivo [40]. DS-epi1 is
350
also highly expressed in esophageal squamous cell carcinoma cells, which exhibit a four to
351
five-fold overexpression in enzyme activity compared with healthy tissue. In this tumor type,
352
DS-epi1 was localized both in the stroma surrounding the tumor and in the tumor cells, and
353
high DS-epi1 expression was also shown to be correlated with increased DS production [41].
354
Other studies have demonstrated that the overexpression of DS in esophageal squamous cell
355
carcinoma cells acts synergistically with HGF by stimulating tumoral migration and invasion.
356
This effect may be the result of the co-receptor abilities of DS with HGF and its respective
357
cellular receptor (c-Met), potentiating the growth factor’s effects [42].
358
Curiously, in our work, MCF7 cells presented higher DS-epi1 protein expression
359
levels than the other cell lines, although this effect was expected for the SKBR3m cell line
360
due to the higher gene expression of this enzyme in this specific cell line. The MCF7 and
361
SKBR3m cell lines both showed significantly increased expression levels of the decorin PG
362
core protein, as detected by flow cytometry.
363
We also examined DS-epi1 localization in SKBR3m, MCF7, and MDA-MB-231 cell
364
lines, using immunofluorescence assays with specific antibodies and confocal microscopy.
365
The SKBR3m cell line was the only cell line to show DS-epi1 content in the perinuclear
366
compartment, suggesting a location to the Golgi apparatus. This localization was further
367
confirmed by the immunofluorescence co-localization assay between the DS-epi1 enzyme and
368
Lectin-HPA (Helix pomatia agglutinin), a specific cellular marker for the Golgi apparatus.
18 369
The MCF7 and MDA-MB-231 cell lines showed cytoplasmic localization of the enzyme,
370
which could explain the increased detection of DS-epi1 by flow cytometry and quantitative
371
immunofluorescence.
372
From the literature, we know that the biosynthesis of GAGs, such as CS, occurs in the
373
endoplasmic reticulum and Golgi apparatus, starting with the addition of a tetrasaccharide and
374
followed by the consecutive addition of hexosamine and a non-nitrogen sugar. For the
375
formation of CS, these sugars correspond with N-acetylgalactosamine and glucuronic acid,
376
respectively. The formation of DS chains involves the conversion of GluA in CS chains to
377
IdoA, through the activity of the DS-epi1 enzyme [21-23]. Active DS-epi1 requires an
378
optimal pH of 5.5 and the presence of divalent cations, especially Mn²⁺ and the weakly
379
activating Ca²⁺ and Mg²⁺ cations. These conditions can be found within the Golgi apparatus,
380
which favors maximal enzyme activity in this organelle [43]. Nevertheless, previous studies
381
have observed that a mere change of 0.2–0.4 pH units in the Golgi luminal pH may promote
382
the mislocalization of several Golgi enzymes, such as α(2,3)-sialyltransferase, to endosomal
383
compartments [44]. Other glycosylation enzymes have also faced similar mislocalization to
384
endosomes or the plasma membrane in similar conditions [45, 46]. MCF7 has been shown to
385
be unable to acidify the Golgi lumen, leading to its alkalinization and mislocalization of
386
several of this organelle’s resident enzymes; a context also investigated in selected colorectal
387
cell lines (e.g., HT-29, SW-48, T84 and Caco-2) [47, 48]. Although there have been no
388
researches regarding how DS-epi1 localization may be affected by alkalinization of the Golgi
389
Apparatus, it is of our belief that certain cancer cell lines which may face it, may lose the
390
epimerization process related to DS synthesis, as suggested here.
391
Among the cell lines examined, SKBR3m was the only cell line containing DS after 35
392
the metabolic labeling of
S-GAGs and characterization with chondroitinase enzymes. As
393
mentioned earlier, DS is implicated in various cancer-promoting processes, such as cell
19 394
proliferation and metastasis. Our data showed increased SKBR3m cell growth, which differed
395
from the other cell lines analyzed, leading us to believe that this phenomenon could be
396
associated with the presence of DS.
397
Our results showed higher gene expression levels for DS-epi1 in SKBR3m cells and
398
the specific subcellular localization of the enzyme to the Golgi apparatus and the presence of
399
35
400
pattern. Our results showed that the localization of DS-epi1 in the Golgi apparatus is of great
401
importance for DS synthesis in breast cancer cell lines, suggesting that in cells where this
402
localization was not observed (e.g., cytoplasmic localization), the formation of DS chains
403
would be compromised. Thus, we suggest that the synthesis of DS could be associated with
404
breast cancer cell growth. The investigation of SKBR3 cells in the present study, which
405
resulted in the identification of the modified cell line, was fundamental to these results. The
406
changes in increased growth, combined with the enhanced synthesis of DS, a compound that
407
has been directly associated with oncogenicity, presents the opportunity to study interactions
408
between DS and critical proteins in the tumor microenvironment (e.g., growth factors,
409
chemokines, and cytokines), and we believe that DS deserves better characterization. The
410
importance of this study is to present a suitable model for breast cancer tumorigenesis and to
411
investigate new therapeutic targets that could be related to the “switch” in DS synthesis
412
observed in this study.
413
CONCLUSION
S-DS in these cells, which also demonstrated a more pronounced cellular proliferation
414
Our results showed that, in SKBR3m cells, the Golgi localization of DS-epi1 was a
415
determinant for DS synthesis. In the MCF7 and MDA-MB-231 cell lines, the cytoplasmic
416
localization of DS-epi1 may be associated with the lack of DS synthesis. However, the
417
decorin PG core protein was still synthesized, especially in SKBR3m and MCF7 cells.
20 418
Moreover, SKBR3m cells were the most viable cell line, and the presence of DS in this cell
419
line may contribute to the proliferative aspects in this particular in vitro model.
420
CONFLICT OF INTEREST
421
422
The authors report no conflict of interest in the content presented in this article.
ACKNOWLEDGMENTS
423
The authors acknowledge Caroline Z. Romera and Elizabeth N. Kanashiro
424
(INFAR/UNIFESP, Brazil) for their confocal technical assistance.
425
FUNDING
426
This work was supported by Fundação de Amparo à Pesquisa do Estado de São
427
Paulo (FAPESP, grant number 2016/18066-6 to LT), Conselho Nacional de Desenvolvimento
428
Científico e Tecnológico (CNPq, fellowship to EGX) and, in part, by the Coordenação de
429
Aperfeiçoamento de Pessoal de Nível Superior — Brasil (CAPES) — Finance Code 001,
430
including a fellowship to EL.
431
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25 520
FIGURE CAPTIONS
521
Figure 1. SKBR3m cells grow faster than MDA-MB-231 and MCF7 cells. The MTT assay
522
was performed at 24, 48, 72, and 96 h. The sample size was n = 16. Data were analyzed by
523
two-way ANOVA with Dunnett’s test. In this particular graph, data is displayed as mean ±
524
standard deviation (SD). * p < 0.05 vs. MDA-MB-231 and MCF7; **** p < 0.0001 vs. MDA-
525
MB-231 and MCF7.
526
Figure 2. The variant SKBR3m grows faster than SKBR3a. The MTT assay was
527
performed at 24, 48, and 72 h. For each time point, an unpaired Student’s t-test was applied to
528
compare OD570 scores between variants. The sample size was n = 24. In this particular graph,
529
data is displayed as mean ± SD. A linear fit was obtained for the variant SKBR3a (R2 =
530
0.928), and an exponential curve was fitted for SKBR3m (R2 = 0.934). * p < 0.05;**** p <
531
0.0001 vs. SKBR3 variants.
532
Figure 3. SKBR3m, and not SKBR3a, synthesizes DS and displays a GAG profile in the
533
cellular medium. (A) For each SKBR3 variant, the
534
extract (C) or medium (M), and after electrophoresis, the CS, DS, and HS contents were
535
analyzed. (B) Each GAG content was quantified and normalized against the total protein
536
quantity in the cell. No GAGs were detected in the medium from SKBR3a cells.
537
Figure 4. SKBR3m cells have a higher gene expression of DS-epi1 and decorin, in
538
comparison to MCF7 and MDA-MB-231 cells. All genes were assessed by RT-qPCR, in
539
which the sample size was n = 5–6. Data were analyzed by two-way ANOVA, with Dunnett’s
540
test. **** p < 0.0001 SKBR3m vs. MCF7; #### p < 0.0001 SBR3 vs. MDA-MB-231; and
541
$$$$ p < 0.0001 MCF7 vs. MDA-MB-231.
542
Figure 5. Concerning the protein expression, MCF7 displays higher DS-epi1 expression,
543
although both MCF7 and SKBR3m cells have high expression of decorin. The flow
35
S-GAGs were assessed in the cell
26 544
cytometry assay was used to analyze the expression profiles of DS-epi1, HS-epi, and decorin
545
proteoglycan. The sample size was n = 4–8. (A) Data were analyzed by two-way ANOVA,
546
with the Holm Šidák test. * p < 0.05; **** p < 0.0001 vs. MDA-MB-231. (B) The histograms
547
show cell population distributions in the flow cytometry experiment, in which unfilled
548
histograms are negative controls from unstained samples.
549
Figure 6. MCF7 and SKBR3m have a higher population of cells that both express DS-
550
epi1 and decorin. The flow cytometry assay analyzed DS-epi1, HS-epi, and decorin
551
proteoglycan expression. Decorin proteoglycan was stained with Alexa 633 and is shown in
552
the y-axis, and either DS-epi1 or HS-epi was labeled with Alexa 488 and is displayed on the
553
x-axis. In red, a representative experimental sample is shown for each cell line; and in blue, a
554
negative control.
555
Figure 7. SKBR3m has a more perinuclear localization of DS-epi1, as opposed to the
556
cytoplasmic display in MDA-MB-231 and MCF7. The immunofluorescence assay was
557
performed in MDA-MB-231 (a and d), MCF7 (b and e) and SKBR3m (c and f) cells
558
assessing DS-epi-1 (a, b or c) or HS-epi (d, e, or f) protein content. Cells were stained with
559
DAPI (1) or F-actin (3), in addition to DS-epi1 or HS-epi (2) labeling. The composite image
560
(4) is also exhibited. The scale bar represents 200 µm, and 7 to 9 fields were analyzed.
561
Figure 8. Although SKBR3m has a more precise localization of DS-epi1, MCF7 displays
562
a higher quantity of it. The data from the immunofluorescence assay was quantified for DS-
563
epi1 and HS-epi content. The sample size was n = 7–9. Data were analyzed by two-way
564
ANOVA, with the Holm Šidák test. ** p < 0.01 vs. MDA-MB-231; ### p < 0.001 vs. MCF7.
565
Figure 9. SKBR3m cells have a higher Golgi colocalization of DS-epi1 than MCF7 and
566
MDA-MB-231. (A) Cells were imaged for DS-epi1 (2) and a Golgi apparatus cellular marker,
567
(lectin HPA, 3). The cells were also stained with DAPI (1). All channels were merged (4), and
27 568
the scale bar represents 50 µm. (B) The representatives' histograms display the colocalization
569
between pixels from the red channel (lectin HPA) and the green channel (DS-epi1) for each
570
tested cell line. (C) The Manders’ Overlap Coefficient (MOC) indicates the degree of
571
colocalization between DS-epi1 with the lectin HPA (i.e., the Golgi marker) for each tested
572
cell line. Data were analyzed with multiple Student’s t-tests. * p < 0.05, **** p < 0.0001 vs.
573
SKBR3m.
574
Figure. 10. Only SKBR3m synthesize DS when compared to MDA-MB-231 and MCF7
575
cells. The assay was performed by labeling GAGs with 35S, purifying them with ethanol, and
576
proteolytically degrading the PG core protein with alcalase. The products were then digested
577
with chondroitinase AC or ABC and separated by electrophoretic mobility in agarose gels in
578
PDA buffer. Only SKBR3m cells displayed DS content, differing from the other cell lines that
579
only contained HS and CS (not shown).
HIGHLIGHTS • • • •
A SKBR3 cell line from a high passage (SKBR3m) is highly proliferative in comparison to MCF7 and MDA-MB-231 cells; The SKBR3m cells were the only to synthesize dermatan sulfate; Dermatan sulfate epimerase 1 (DS-epi1) was found in the Golgi Apparatus only in SKBR3m cells; MCF7 and MDA-MB-231 cells presented DS-epi1 mislocalized.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: