ZEB1

E-Cadherin

Actin

PanC-1

Starvation - + + + + TGFβ1 - - - + +Panobinostat (25nM) - - + - +

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

AbstractHDAC 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 E-Cadherin 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 E-Cadherin 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 E-Cadherin 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 co-activator 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 E-Cadherin induction.

ZEB1 is involved in HDACi-induced E-Cadherin induction:

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, E-Cadherin 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.

Panobinostat reverses EMT in vivo:

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).

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.

Histone deacetylase (HDAC) inhibition is known to inhibit the Epithelial-to-Mesenchymal 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

Conclusion:HDAC inhibitors inhibit epithelial to mesenchymal transition in pancreatic cancer by inhibiting the expression of ZEB1

Results

Methods

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 5: SNAIL1 and SNAIL2 are not involved in HDACi-induced E-Cadherin expression

Around 1X106 exponentially 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.

Figure 6: ZEB1 overexpression abrogates HDACi-induced E-Cadherin up regulation

Figure 8: HDAC inhibitors suppress tumor growth and induce E-Cadherin expression in vivo

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 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.

PanC-1 BxPC-3

HPAC

Panobinostat (nM) - 25 50 - - - -Trichostatin A (nM) - - - 100 200 - - Veronistat (µM) - - - - - 1 2

Panobinostat (nM) - 5 10 - - - -Trichostatin A (nM) - - - 100 200 - - Veronistat (µM) - - - - - 1 2

Panobinostat (nM) - 5 10 - - - -Trichostatin A (nM) - - - 100 200 - - Veronistat (µM) - - - - - 1 2

E-Cadherin

Cytokeratin 18

Actin

E-Cadherin

Cytokeratin 18

Actin

E-Cadherin

Cytokeratin 18

Actin

Figure 4: Effect of HDAC inhibitor on E-Cadherin transcriptional repressors

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.

SNAIL1

SNAIL2

TWIST

ZEB1

SNAIL1

SNAIL2

TWIST

ZEB1

Panobinostat (nM) - 25 50 - - - -Trichostatin A (nM) - - - 100 200 - - Veronistat (µM) - - - - - 1 2

Panobinostat (nM) - 25 50 - - - -Trichostatin A (nM) - - - 100 200 - - Veronistat (µM) - - - - - 1 2

ActinActin

PanC-1 BxPC-3

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 3: Immunoflorescence of HDAC inhibitor induced E-Cadherin expression

Pan

C-1

Control Panobinostat (25nM)

E-Cadherin immunoflorescence

Figure 2: HDAC inhibitors induce E-Cadherin expression transcriptionally

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.

502 bp

PanC-1, RT-PCR

Panobinostat (25nM) - + - - Veronistat (1µM) - - - +

349 bp

PanC-1, IP: SNAIL1

HDAC1

SNAIL1

Animal tumors

Control Panobinostat

E-Cadherin

ZEB1

Actin

Panobinostat (nM) 0 25

A B

A

B

C

SNAIL2

E-Cadherin

Actin

Empty vector - + - - - SNAIL2 ove. exp. - - - + +Panobinostat (25nM) - - + - +