HDAC inhibition reverses epithelial to mesenchymal transition through modulation of ZEB1
Srinivas Reddy Boreddy, Prabodh K. Kandala, Parul Gupta, Sanjay K. Srivastava.
Department of Biomedical Sciences & Cancer Biology Center, Texas Tech University Health Sciences Center, School of Pharmacy, Amarillo Texas 79106
Abstract
Histone deacetylase (HDAC) inhibition is known to inhibit the Epithelial-toMesenchymal Transition (EMT) in various cancer models by suppressing the
acetylation of histone H3 and H4 of E-Cadherin promoter. However, the exact
mechanism of EMT inhibition by HDAC inhibition is not known. In the present study,
using various HDAC inhibitors such as Vorinostat (SAHA), Tricostatin A (TSA) and
Panobinostat (LBH-589), we explored the molecular mechanisms of EMT inhibition in
pancreatic tumor cells in vitro and in vivo. The expression of epithelial markers such as
E-Cadherin and cytokeratin 18 were significantly up regulated in PanC-1, BxPC-3 and
HPAC cells treated with HDAC inhibitors, whereas the expression of mesenchymal
marker vimentin decreased. Morphology of the cells also changed indicating that
HDAC inhibitors suppressed EMT in pancreatic cancer cells. To determine whether
HDAC inhibition correlates with the down-regulation of key transcriptional suppressors
of E-Cadherin such as SNAIL1, SNAIL2, TWIST and ZEB, cells were treated with
panobinostat. To our surprise, SNAIL1 expression was drastically increased, whereas
expression of SNAIL2, TWIST and ZEB were decreased by HDAC inhibition in all the
cell lines. Since, it has been previously shown that HDAC activity is required to
suppress E-Cadherin expression by its repressors, we wanted to see whether SNAIL1
also requires HDAC activity to repress E-Cadherin expression in our model. HDAC1/2
was co-immunoprecipitated with SNAIL1. Our results revealed that this interaction was
more pronounced in control cells as compared to HDAC inhibitors treated cells. These
results suggest that although SNAIL1 expression was significantly increased by HDAC
inhibition, it remained ineffective in E-Cadherin promoter. To evaluate other repressors,
PanC-1 cells were transfected with SNAIL2 and ZEB1 and treated with HDAC
inhibitors. Interestingly, our results show that SNAIL2 overexpression was unable to
abrogate increase in E-Cadherin by HDAC inhibition. However, ZEB1 overexpression
completely blocked the induction of HDAC-mediated E-Cadherin expression. To
validate in vitro observations in an in vivo model, BxPC-3 cells were subcutaneously
implanted in athymic nude mice. When the tumors reached about 70mm3, 20mg/Kg
panobinostat was administered intraperitonealy three times a week while control group
received vehicle alone. After 4 weeks of treatment, the growth of pancreatic tumors was
significantly suppressed in panabinostat treated mice. Moreover, the tumors from
panabinostat treated mice showed an increase in E-Cadherin and decrease in ZEB
expression. The expression of other E-Cadherin repressors SNAIL1 and SNAIL2 were
also down regulated. In conclusion, our in vitro and in vivo results clearly show that
increase in E-Cadherin by HDAC inhibition was due to the down regulation of ZEB1.
[Supported in part by R01 grants CA129038 and CA106953 (to S.K.S) awarded by the
National Cancer Institute].
Figure 1: HDAC inhibitors induce E-Cadherin expression in
pancreatic cancer cells
PanC-1
A
PanC-1, IP: SNAIL1
B
E-Cadherin
E-Cadherin
Cytokeratin 18
Cytokeratin 18
Actin
Actin
Panobinostat (nM)
Trichostatin A (nM)
Veronistat (µM)
-
25 50
- - -
100
-
200
-
1
SNAIL2
HDAC1
Panobinostat (nM)
Trichostatin A (nM)
Veronistat (µM)
2
-
5 10
- - -
100 200
-
1
E-Cadherin
Actin
2
Empty vector SNAIL2 ove. exp. Panobinostat (25nM) -
HPAC
+
-
+
+
-
+
+
SNAIL1
E-Cadherin
Panobinostat (nM) 0
Cytokeratin 18
Actin
Panobinostat (nM)
Trichostatin A (nM)
Veronistat (µM)
-
5 10
- - -
100
-
200
-
1
25
PanC-1 cells were treated with panobinostat for 72h and whole cell lysates were isolated. A). Equal amount of SNAIL1 protein was
immunoprecipitated in control and treated cells and immunoblotted for HDAC1. B). SNAIL2 was overexpressed in PanC-1 cells using
FuGene 6, according to manufacturer’s instructions, and treated with panobinostat for 72h. Eual amount of protein from control and treated
cells was subjected to western blot.
2
Figure 6: ZEB1 overexpression abrogates HDACi-induced ECadherin up regulation
Pancreatic cancer cells were seeded in a 6-well plate and treated with HDAC inhibitors for 72h. Cells were collected and whole cell lysates
were analyzed for epithelial markers’ expression by western blot. Actin was used as a loading control.
Figure 2: HDAC inhibitors induce E-Cadherin expression
transcriptionally
PanC-1
ZEB1
PanC-1, RT-PCR
E-Cadherin
502 bp
Actin
Starvation
TGFβ1
Panobinostat (25nM)
-
+
-
+
+
+
+
-
+
+
+
349 bp
Panobinostat (25nM)
Veronistat (1µM)
-
+
-
-
PanC-1 cells were pre-treated with TGFβ1 for 1h and co-treated with panobinostat for additional 72h. Cells were collected and subjected to
western blot. Actin was used a loading control.
+
PanC-1 cells were treated with panobinostat and SAHA for 72h and total RNA was isolated by Trizol method. RNA was subjected RT-PCR using
Verso 1-step PCR kit (Thermo Scientific) to analyze the levels of E-Cadherin RNA transcripts. GAPDH was used as a loading control.
Figure 3: Immunoflorescence of HDAC inhibitor induced ECadherin expression
Figure 8: HDAC inhibitors suppress tumor growth and induce
E-Cadherin expression in vivo
A
E-Cadherin immunoflorescence
Methods
Control
Panobinostat (25nM)
HDAC inhibitors up regulates E-Cadherin expression in pancreatic cancer
cells:
To see whether HDAC inhibitors inhibit epithelial to mesenchymal transition
(EMT) by up regulating E-Cadherin expression, BxPC-3, HPAC and PanC-1 cells
were treated with different concentrations of panobinostat, trichostatin A and
veronistat. Cells were collected and E-Cadherin expression was evaluated by
western blot. As shown in Fig. 1, epithelial markers, such as E-Cadherin and
cytokeratin 18 were up regulated in HDAC inhibitor treated cells, as compared to
control cells.
HDAC inhibitors transcriptionally up regulates E-Cadherin expression:
Next we wanted to know whether HDAC inhibitors transcriptionally up regulate ECadherin expression in pancreatic cancer cells. To test this cells were treated with
HDAC inhibitors and RT-PCR analysis was performed. Interestingly, all the HDAC
inhibitors significantly increased mRNA levels of E-Cadherin, indicating that ECadherin was transcriptionally up regulated (Fig.2).
Evaluation of E-Cadherin by Immunoflorescence:
E-Cadherin up regulation by HDAC inhibitors was further confirmed by
immunoflorescence. PanC-1 cells were treated with panobinostat for 72h and cells
were immunostained for E-Cadherin expression. Similar to our western blot
analysis, as compared to control cells, panobinostat treated cells showed intense
red color staining indicating E-Cadherin (Fig.3).
HDAC inhibitors suppress E-Cadherin transcriptional repressors:
Since, E-Cadherin expression is tightly regulated by its transcriptional repressors,
such as SNAIL1, SNAIL2, TWIST and ZEB1 we wanted to see the effect of
HDAC inhibitors on these repressors. Cells were treated with various HDAC
inhibitors and whole lysates were subjected to western blotting. Surprisingly,
SNAIL1 was up regulated and SNAIL2 and ZEB1 were down regulated, whereas
TWIST was moderately altered in HDACi-treated pancreatic cancer cells (Fig.4).
SNAIL1 and SNAIL2 are not involved in HDAC inhibitor induced ECadherin expression:
Since we observed down regulation of all E-Cadherin repressors, except SNAIL1
by HDACi treatment, we next wanted to see which one of these repressors was
critically involved in HDACi-induced E-Cadherin up regulation. We also wondered
how E-Cadherin is induced in spite of SNAIL1 up regulation by HDACi treatment.
We hypothesized that although SNAIL1 was up regulated by HDACi its coactivator HDAC1/2 was down regulated by HDACi (Fig.5A). Hence SNAIL1 was
ineffective on E-Cadherin promoter. Furthermore, SNAIL2 overexpression could
not abrogate HDACi-induced E-Cadherin induction (Fig.5B). Taken together, our
results indicate that SNAIL1 and SNAIL2 were not involved in HDACi-induced ECadherin induction.
ZEB1 is involved in HDACi-induced E-Cadherin induction:
PanC-1
Cell culture: PanC-1 and HPAC cells were cultured in DMEM medium, whereas BxPC-3
cells were cultured in RPMI supplemented with 10% FBS and antibiotics.
RT-PCR: Pancreatic cancer cells were treated with HDAC inhibitors (HDACi) and total RNA
was isolated by Trizol method. RNA was subjected to RT-PCR analysis using Verso 1-step
PCR kit (Thermo scientific, IL) for E-Cadherin expression.
Immunoflorescence: PanC-1 cells were treated with panobinostat for 72h and fixed with
methanol:acetone (50:50, -200C). Cells were probed with E-Cadherin and actin primary
antibody followed by Alexa Fluor-594/488 secondary antibody. Cells were photographed
under florescence microscopy.
Transfections: All the transfections were performed using FuGene 6 reagent (Roche, IN),
according to manufacturer's instructions.
Western blot analysis: Cells were exposed to varying concentrations of deguelin for the
indicated time periods. Cell lysates were subjected to SDS-PAGE and the proteins were
transferred onto PVDF membrane and incubated overnight with the desired primary antibody.
Subsequently, the membrane was incubated with appropriate secondary antibody, and the
immunoreactive bands were visualized using enhanced chemiluminescence kit (NEN Life
Science Products, Boston, MA) according to the manufacturer’s instructions. The same
membrane was re-probed with the antibody against actin (1:50,000 dilution) and used as an
internal control for equal protein loading after stripping with stripping buffer.
In vivo studies: All the experiments involving animals were approved by the Institutional
Animal Care and Use Committee (IACUC). Twenty 4-6 week old female athymic nude mice
(Charles River, Wilmington, MA) were injected with 1X106 exponentially growing BxPC-3
cells on the both flanks of nude mice. After 7 days mice were randamized and treated group
received 20mg/kg panobinostat every 72h for 42 days. Tumors were measured thrice a week
using vernier calipers. At the end of the experiment animals were euthanized and tumors were
excised. Control and treated tumors were lysed and subjected to western blot.
Statistical analysis: All the statistical analysis was performed using Prism 4.0 (GraphPad
software Inc., San Diego, CA). Results were expressed as mean ± SD of the at least three
independent experiments. Data was analyzed by Student’s t-test or one way ANOVA followed
by Bonferroni’s post hoc analysis for multiple comparisons. Differences were considered
statistically significant at p<0.05.
BxPC-3
Figure 5: SNAIL1 and SNAIL2 are not involved in HDACiinduced E-Cadherin expression
Results
To evaluate whether ZEB1 is involved in HDACi-induced E-Cadherin expression
PanC-1 cells were pre-treated with TGFβ1 for 1h and followed by treatment with
panobinostat for additional 72h. Our results show that TGFβ1 significantly induced
ZEB1 expression. However when these cells were treated with panobinostat, ECadherin induction was abrogated (Fig.6), indicating the involvement of ZEB1 in
HDACi-induced induction of E-Cadherin. Further studies are required to confirm
this observation.
PanC-1 cells were treated with panobinostat (25nM) for 72h and cells were subjected to immunoflorescence to visualize E-Cadherin (red) and
actin (green).
Figure
4:
Effect of HDAC inhibitor
transcriptional repressors
on
PanC-1
Panobinostat reverses EMT in vivo:
C
E-Cadherin
Animal tumors
Control
Panobinostat
BxPC-3
SNAIL1
SNAIL2
TWIST
ZEB1
SNAIL1
E-Cadherin
SNAIL2
ZEB1
TWIST
Actin
We were curious to see whether panobinostat could reverse EMT in vivo. To test
this BxPC-3 cells were injected into the flanks of nude mice and treated with
20mg/kg panobinostat over the period of 42 days. Tumor volume was measured by
vernier calipers thrice a week. Our results show that panobinostat suppressed the
growth of BxPC-3 xenografts by 36%, as compared to control animals
(Fig.7A&B). At the end of the experiment tumors were excised and subjected to
western blot. Our results show that E-Cadherin expression was significantly
increased, whereas ZEB1 expression was decreased in the tumors of panobinostat
treated mice, as compared to control mice (Fig.7C).
ZEB1
Actin
Panobinostat (nM)
Trichostatin A (nM)
Veronistat (µM)
B
Actin
-
25 50
- - -
100
-
200
-
1
2
Panobinostat (nM)
Trichostatin A (nM)
Veronistat (µM)
-
25 50
- - -
100
-
200
-
1
2
PanC-1 and HPAC cells were treated with HDCi for 72h and whole cell lysates were collected. Equal protein from control and treated was
subjected to SDS-PAGE followed by western blot to analyze the expression of E-Cadherin repressors. Actin was used as a loading control.
1X106 exponentially
Around
growing BxPC-3 cells were injected on the both flanks of the nu-nu mice. After 7 days animals were randomized
and treated group received 20mg/kg panobinostat at every 72h. A).Tumors were measured twice a week using vernier calipers and
presented as line diagram. B). Tumor growth suppression by panobinostat by the end of the experiment. C). At the end of the experiment
animals were euthanized, tumors were carefully excised and snap frozen in liquid nitrogen. Tumor were lysed and equal amount of protein
was subjected to western blot. *=P<0.05 statistically significant compared to control animals.
Conclusion:
HDAC inhibitors inhibit epithelial to mesenchymal transition
in pancreatic cancer by inhibiting the expression of ZEB1