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Activin A inhibits the proliferation of ER?+?ve and ER-ve breast cancer cells

Previous reports have shown that proliferation of ER?+?ve cells is inhibited by activin
14], but the effect of activin on proliferation of ER-ve cell lines is less clear 11]. In order to compare the effect of activin and follistatin on proliferation in ER-ve
and ER?+?ve cell lines, a time course and dose response MTS assay was performed. Both
the ER-ve (MDA-MB-231) and ER?+?ve (MCF7) cells showed a significant decrease in proliferation
compared to control following addition of activin A on days 1 and 3 (Figure 2A?+?B) which was lost by day 5 (data not shown). There was a significant dose-dependent
inhibition of proliferation with increasing doses of activin A in both cell lines.
These results show that both ER-ve and ER?+?ve cell lines are responsive to the growth
inhibitory effect of activin A. To confirm the decrease in proliferation was occurring
in an activin dependent manner in ER-ve cell lines given the controversy in the literature,
MDA-MB-231 cells were treated with activin 6000 pg/ml +/? an ALK4/5 inhibitor SB-431-542
(10 ?M/l) for 72 hours. The ALK4/5 inhibitor will prevent ActRII dimerising with its
type I receptor ALK4. MDA-MB-231 cells showed less inhibition of proliferation with
addition of the ALK4/5 inhibitor to activin (mean % change in absorbance from control
Activin; ?11%, Activin?+?ALK4/5 inhibitor ?3.4%) confirming the changes in proliferation
in ER-ve cells was activin-dependent (data not shown).

Figure 2. Activin inhibits breast cancer cell proliferation. MDA-MB ?231 (A?+?C) and MCF7 (B?+?D) cells were treated with increasing doses of activin in a timecourse experiment (A?+?B), or with recombinant activin (6000 pg/ml) +/? follistatin (64,000 pg/ml) for 72 hours
(C?+?D). 20 ?l of MTS solution was added 4 hours prior to the final time points to evaluate
absorbance of treated cells relative to control untreated cells. Data represents mean?+?SEM
of 8 replicates and 5 repeats. Wilcoxon Signed-Rank test for significance, *p value
0.05, NS not significant.

Follistatin impairs the inhibition of proliferation induced by activin A

Follistatin is reported to negate the anti-proliferative effect of activin 12]. In order to evaluate the effect of follistatin in ER-ve (MDA-MB-231) and ER?+?ve
(MCF7) cells they were treated with 6000 pg/ml of activin A in the presence or absence
of follistatin (64,000 pg/ml) for 72 hours (activin concentrations were chosen to
replicate inter-tumoural levels of activin in breast tumours 15]). The significant inhibitory effect of activin A on cell proliferation was negated
in the presence of follistatin in MDA-MB-231 cells (mean % change from control; Activin
?6% [SEM 0.92], Activin?+?follistatin +2.6% [SEM 1.9]). In MCF7 cells a similar, but
non-significant, trend was also seen (mean % change from control; Activin ?3.2% [SEM
1.3], Activin?+?follistatin ?0.02% [SEM 2.4]) (Figure 2C?+?D). These data provided further indication that ER-ve cell lines are sensitive
to the growth inhibitory effects of activin A and that this effect is inhibited by
follistatin.

ER-ve cells secrete more activin A and follistatin than ER?+?ve cells

The quantity of activin A and follistatin secreted from ER-ve (MDA-MB-231 and MDA-MB-436),
and ER?+?ve (MCF7 and T47D) cells was determined by ELISA. As shown in Figure 3A, both ER-ve cell lines secreted significant levels of activin A (MDA-MB-231?=?561 pg/ml
[SEM 104.9] p value?=?0.0034, MDA-MB-436?=?430 pg/ml [SEM 73.8] p value?=?0.0436).
Follistatin was detectable in the medium from all cell lines, although the levels
were much lower in ER?+?ve cell lines (mean level pg/ml; MCF7?=?200, T47D?=?108) compared
to ER-ve cell lines (mean level pg/ml; MDA-MB-231?=?7224, MDA-MB-436?=?1704 Figure 3B). These results suggest that ER-ve cells could be more dependent on activin A for
regulation of cell growth than ER?+?ve cells, and may utilise secretion of follistatin
as a mechanism of escape from the anti-proliferative effects of activin A. There is
no clear evidence to indicate why tumor cells secrete a growth inhibitor such as activin
16], suggesting it may be have alternative functions that the cells escape from by alternative
mechanisms i.e. secretion of inhibitors or down regulation of receptors.

Figure 3. Activin and follistatin secretion from ER- breast cancer cell lines and ER?+?ve breast
cancer cell lines. 1×10⁵ MDA-MB-231 or MDA-MB-436 (ER-ve) and 4×10⁵ MCF7 or T47D (ER?+?ve) cell lines were plated in 6 well plates and levels of Activin
(A) and follistatin (B) in the medium determined by ELISA at 24 and 48 hours. Data represents 3 replicates
and 3 repeats. Mann Whitney test for significance comparing wells with cells to media
alone (no cells), *p value 0.05. MDL?=?below assay minimum detection limit.

Zoledronic acid differentially affects proliferation of breast cancer cell lines according
to ER status

To evaluate if zoledronic acid could affect proliferation of ER?+?ve and ER-ve breast
cancer cell lines MDA-MB-231, MDA-MB-436, T47D and MCF7 cells were treated with 50 ?M
ZOL or medium control for 48 hours and live cell count performed with trypan blue.
ZOL significantly inhibited proliferation of both ER-ve cell lines compared to control
(Cell count x10^5; MDA-MB-231 control 3.4 [SEM 0.79], ZOL 1.6 [SEM 0.25] p value 0.0009, MDA-MB-436
control 6.9 [SEM 0.33] ZOL 5.3 [SEM 0.14] p value 0.0043), but did not significantly
alter proliferation of the ER?+?ve cell lines MCF7 and T47D (Figure 4).

Figure 4. Effects of zoledronic acid on proliferation of ER?+?ve and ER-ve cell lines. 1×10⁵ MDA-MB-231, MDA-MB-436 (ER-ve),MCF7 or T47D (ER?+?ve) cell lines were plated in 6
well plates. Cell were treated for 48 hours with medium +/? 50 ?M ZOL. At 48 hours
viable cell count was performed using trypan blue. Data represents 3 replicates and
3 repeats. Mann Whitney test for significance comparing control with ZOL treated,
NS not significant, **p value??0.005, ***p value 0.0005.

Zoledronic acid decreases follistatin secretion from ER-ve cell lines only

To investigate if ZOL could affect the secretion of activin A and/or follistatin from
MDA-MB-231 and MCF7 cells, both cell lines were exposed to medium +/? 25 ?M/50 ?M
ZOL for 48 hours. Activin A secretion was unaffected by ZOL in either cell line. In
contrast, follistatin secretion was significantly decreased in MDA-MB-231 cells after
exposure to ZOL (control 23378 pg/ml [SEM 5259], ZOL 9987 pg/ml [SEM 2871], p value
=0.0012), and also fell in MDA-MB-436 cells (control 1928 pg/ml [SEM 188], ZOL 1592 pg/ml
[SEM 65] p value 0.07), but did not change in MCF7 (Figure 5) or T47D cells (data not shown). We hypothesise that the biological activity of activin
A depends on the ratio of activin A to follistatin in the tumour microenvironment.
The literature reports a 4:1 molar ratio of follistatin:activin would neutralize activin
17]. ZOL reduced the molar ratio of follistatin:activin secreted by MDA-MB-231 cells
(control ratio 14:1, ZOL ratio 4:1) (Figure 5B), compared to minimal change in MCF7 cells (control ratio 3:1, ZOL ratio 4:1) (Figure 5D) indicating ZOL has a more noticeable effect on the follistatin:activin ratio in
ER-ve cell lines.

Figure 5. Effects of zoledronic acid on follistatin secretion and follistatin:activin ratio. MDA-MB-231 (A) and MCF7 (C) cells were treated with medium alone, 25 ?M or 50 ?M ZOL for 48 hours and levels
of secreted activin and follistatin measured by ELISA. Molar ratio of follistatin:activin
(B?+?D) was calculated by converting mean quantity of secreted protein per million cells
(pg/ml) to pmol/l by dividing by the molecular weight of each protein, and then expressed
as a ratio. A molar ratio of 4 (dashed line) represents the level at which activin
is neutralised by follistatin: an excess of follistatin:activin increases tumour growth
(above dashed line). Data represents mean?+?SEM of 3 replicates and 3 repeats, *=?p
value 0.05, NS not significant.

To evaluate the effects of a short (clinically achievable) exposure of ZOL on follistatin
secretion in ER-ve cell lines, MDA-MB-231 and MDA-MB-436 cells were treated with 50 ?M
ZOL for 4 hours, followed by 44 hours incubation in drug-free medium. The secretion
of follistatin was significantly decreased in both cell lines by a 4-hour pulse of
ZOL (mean follistatin pg/ml, MDA-MB-231; control?=?17551 [SEM 847], ZOL?=?6106 [SEM
1315] p value =0.0015. MDA-MB-436; control?=?3209 [SEM 236], ZOL =1667 [SEM 116] p
value?=?0.001) (Figure 6A). These data show that even a short exposure to ZOL decreases follistatin secretion
from ER-ve cell lines.

Figure 6. Effects of pulsed zoledronic acid on follistatin secretion from ER-ve cell lines.
The difference in follistatin secretion according to ER status is not due to differences
in cellular uptake of the drug. A. MDA-MB-231 and MDA-MB-436 cells were treated for 4 hours with 50 ?M of ZOL followed
by a 44 hour incubation with medium alone or medium alone for 48 hours (CON). Follistatin
levels in the supernatant was removed and processed for ELISA. Data represents mean?+?SEM
of 3 replicates and 3 repeats, **=?p value 0.01, ***=?p value 0.001. B. MDA-MB-231 and MCF7 cells were treated for 48 hours with medium alone (C), GGOH
50 ?M (G), Zol 10 ?M (Z) or both G and Z in combination (ZG). Rap1a antibody (1:200)
used to assess levels of unprelylated protein and GAPDH (1:20,000) used as loading
control.

Both ER?+?ve and ER-ve cell lines take up ZOL in vitro

ZOL increases accumulation of unprenylated small GTPases i.e. Rap1a via inhibition
of the mevalonate pathway 18]. The lack of effect of ZOL on follistatin secretion in the ER?+?ve cells was considered
to possibly reflect a limited drug uptake. To evaluate if the ER?+?ve and ER-ve cell
lines used in this study had a similar levels of ZOL uptake we used western blotting
to compare the accumulation of unprenylated Rap1a (uRap1a, a surrogate marker of ZOL
uptake) in MCF7 and MDA-MB-231 cells treated with ZOL, and if addition of the mevalonate
pathway intermediary, geranylgeraniol (GGOH), could inhibit the accumulation of uRap1a.
Both cell lines had increased levels of uRap1a in response to treatment with ZOL that
was partially reversed by addition of GGOH (Figure 6B). These data suggest that the difference in follistatin secretion between ER-ve
and ER?+?ve cell lines is not due differential cellular uptake of ZOL.

Zoledronic acid reduces intracellular C terminus phosphorylated Smad2

To evaluate if the activin-signaling pathway downstream of surface receptors is affected
by ZOL, localization and intracellular quantity of pSmad2C in MDA-MB-231 and MCF7
cells was assessed. Using an immunofluorescence method, we detected no significant
difference in the percentage of cells with nuclear localization of pSmad2C after treatment
with ZOL compared to control in either cell line (Figure 7A-C). However, MDA-MB-231 cells exposed to CM from ZOL treated cells (containing low
levels of follistatin) showed significantly higher levels of pSmad2C than cells exposed
to CM from medium only treated cells (control?=?0.29 [SEM 0.065], ZOL?=?0.7 [SEM 0.14],
p value 0.0286) (Figure 7D). This effect was not seen in MDA-MB-231 cells exposed directly to 50 ?M ZOL, indicating
that the decreased follistatin levels in CM from ZOL treated cells was responsible
for the increase in intracellular levels of pSmad2C.

Figure 7. Smad2 phosphorylation at both c terminus and linker region is differentially affected
by zoledronic acid according to ER status of breast cancer cells. A?+?B Representative immunofluorescent images of pSmad2C (green) and dapi staining (blue)
in MDA-MB-231 cells (A) and MCF7 cells (B) treated with medium alone (C) or ZOL (Z) for 48 hours. C. Quantification of nuclear localisation of pSmad2C in MDA-MB-231 and MCF7 cells treated
for 48 hours with medium alone (con) or ZOL (50 ?M). Data represents minimum 100 cells
per group, Mann Whitney U test for significance, NS?=?not significant D. Ratio of total cellular quantity of total Smad2/3 to pSmad2/3 in MDA-MB-231 cells
treated for 1 hour with medium alone, ZOL (50 ?M) or conditioned medium (CM) from
MDA-MB-231 cells previously treated with ZOL (50 ?M) or medium (control). Data represents
3 replicates and 3 repeats, Mann Whitney U test for significance, NS?=?not significant,
*p?=?0.05 E?+?F Representative immunofluorescent images of pSmad2L (green) and dapi staining (blue)
in MDA-MB-231 cells (E) and MCF7 cells (F) treated with medium alone (C) or ZOL (Z) for 48 hours. G. Quantification of nuclear localisation of pSmad2L in MDA-MB-231 and MCF7 cells treated
for 48 hours with medium alone (con) or ZOL (50 ?M). Data represents minimum 100 cells
per group, Mann Whitney U test for significance, NS?=?not significant, ***p value
0.001. H. Representative western blots for cellular quantity of pSmad2L and gapdh in MDA-MB-231
cells treated with medium alone (con) or ZOL (50 ?m).

Zoledronic acid decreases nuclear localization of linker phosphorylated Smad2

Whereas pSmad2C is recognized to function as a tumour suppressor in breast cancer
19], pSmad2L may act as a tumour growth promoter 10]. We evaluated if ZOL could affect cellular localization of pSmad2L in MDA-MB-231
and MCF7 cells using immunofluorescence. The percentage of MDA-MB-231 cells with nuclear
localization of pSmad2L was significantly decreased after treatment with ZOL (control?=?50%
[SEM 4.6], ZOL =6.6% [SEM 1.1], p value 0.0001). No significant difference was seen
between ZOL and control in the MCF7 cells (Figure 7E-G). Using western blotting we found that ZOL did not cause a significant alteration
in the total cellular levels of pSmad2L, suggesting that ZOL alters cellular localization
of pSmad2L in MDA-MB-231, but not the total quantity (Figure 7H).

Zoledronic acid decreases follistatin and pSmad2L expression in an ER-ve xenograft
model

In order to validate that ZOL induces changes in follistatin and pSmad2L in ER-ve
tumours in vivo, ER-ve MDA-MB-436 sub-cutaneous tumour sections from mice treated with or without
ZOL (100 ?g/kg, weekly for 6 weeks, equivalent to the 4 mg clinical dose) were evaluated.
Follistatin expression was scored for intensity and area of positive stain. Data were
analysed using average scores from 2 assessors blinded to the treatment groups. No
difference was seen in the intensity of follistatin staining, however, there was a
significant decrease in the tumour area staining positive for follistatin in mice
treated with ZOL compared to saline (Figure 8A-D). The number of cells with mitotic nuclei staining positive for pSmad2L was significantly
lower in tumours from ZOL treated mice compared to saline treated (Figure 8E-F). These results suggest that using the dosing regime described, ZOL can directly
alter expression of both follistatin and pSmad2L in ER-ve subcutaneous tumours in vivo.

Figure 8. Follistatin and pSmad2L expression in MDA-MB-436 xenografts is reduced following zoledronic
acid treatmentin vivo. A. Representative images of follistatin expression in tumours from saline treated mice
at x1.6 magnification (left) and x20 magnification (right). Viable tumour cells (T),
necrotic core of tumours (NC). B Representative images of follistatin expression in tumours from ZOL treated mice.
C?+?D. 20 x 750 ?m² images were scored from two sections per tumour. Images were scored for intensity
of?+?ve stain (C) and area of?+?ve stain (D). Data represents the mean scores?+?SEM. Mann Whitney U test for significance, ***p
value 0.001, NS not significant. E Representative images of pSmad2L expression (black arrows) in saline treated mice
(C) and ZOL treated mice (Z). F 20×750?m² images were scored from two sections per tumour. Number of positive cells were counted
and data represents mean scores?+?SEM. Mann Whitney U test for significance, ***p
value 0.001.