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, 7 (18), 26551-66

The GSK3β Inhibitor BIS I Reverts YAP-dependent EMT Signature in PDAC Cell Lines by Decreasing SMADs Expression Level

Affiliations

The GSK3β Inhibitor BIS I Reverts YAP-dependent EMT Signature in PDAC Cell Lines by Decreasing SMADs Expression Level

Natthakan Thongon et al. Oncotarget.

Abstract

The Yes-associated protein, YAP, is a transcriptional co-activator, mediating the Epithelial to Mesenchymal Transition program in pancreatic ductal adenocarcinoma (PDAC). With the aim to identify compounds that can specifically modulate YAP functionality in PDAC cell lines, we performed a small scale, drug-based screening experiment using YAP cell localization as the read-out. We identified erlotinib as an inducer of YAP cytoplasmic localization, an inhibitor of the TEA luciferase reporter system and the expression of the bona fide YAP target gene, Connective Tissue Growth Factor CTGF. On the other hand, BIS I, an inhibitor of PKCδ and GSK3β, caused YAP accumulation into the nucleus. Activation of β-catenin reporter and interfering experiments show that inhibition of the PKCδ/GSK3β pathway triggers YAP nuclear accumulation inducing YAP/TEAD transcriptional response. Inhibition of GSK3β by BIS I reduced the expression levels of SMADs protein and reduced YAP contribution to EMT. Notably, BIS I reduced proliferation, migration and clonogenicity of PDAC cells in vitro, phenocopying YAP genetic down-regulation. As shown by chromatin immunoprecipitation experiments and YAP over-expressing rescue experiments, BIS I reverted YAP-dependent EMT program by modulating the expression of the YAP target genes E-cadherin, vimentin, CTGF and of the newly identified target, CD133. In conclusion, we identified two different molecules, erlotinib and BIS I, modulating YAP functionality although via different mechanisms of action, with the second one specifically inhibiting the YAP-dependent EMT program in PDAC cell lines.

Keywords: CTGF; EMT; PDAC; YAP; bisindolylmaleimides.

Conflict of interest statement

The authors disclose no conflicts of interest.

Figures

Figure 1
Figure 1. Importance of YAP in PDAC cell lines
A. YAP is expressed in PDAC lines at different levels. Western blot analysis of endogenous level of YAP in PDAC cell lines and qRT-PCR analysis of YAP mRNA expression. The relative intensity of the bands (left) and YAP mRNA level (right) are shown. B. Localization of YAP is regulated by cell density in PK9. PK9 and PANC1 cells were cultured sparsely (LOW) and densely (HIGH) onto glass cover slides (Left panel) and in 96 well-plate (right panel) for 48H. Cells were fixed and nuclei were counterstained with DAPI. The localization of YAP was visualized using a Zeiss Observer Z1 microscope equipped with Apotome module, with a Plan Apochromatic (63X, NA 1.4) objective. Images were acquired using Zen 1.1 (blue edition) imaging software (Zeiss) and assembled with Adobe Photoshop CS3 (Left panel). Quantitative analysis of sub-cellular localization of YAP was quantified using Operetta instrument and Harmony 3.5.2 software. Ratio of YAP Nuc/Cyto is shown. (*p<0.05). C. YAP functional ablation down-regulates CTFG and CYR61 but not AREG and BIRC5 mRNA levels. PANC1 (left) and PK9 cells (right) were stably transduced with a lentiviral vector encoding shRNA targeting YAP or a non-targeting control shRNA (SCR). After stable selection with puromycin, the relative levels of endogenous YAP and its target genes, CTGF, CYR61, AREG and BIRC5 mRNA were measured by qRT-PCR (mean±SD). (**P<0.0, **P<0.01 versus SCR). D. Overexpression of YAP increases CTGF and CYR61 levels in PK9 cells. PK9 and PANC1 cells were transiently transfected with pEGFP-YAP or empty vector (pEGFPN1) for 24H. Overexpression of YAP was confirmed by qRT-PCR analyses. The expression levels of CTGF and CYR61 were then evaluated. E. YAP functional ablation attenuates anchorage-independent growth in soft agar. PANC1 cells were stably transduced with a lentiviral vector encoding shRNA targeting YAP or a non-targeting control shRNA (SCR). These clones (1.5×104 cells) were seeded in 0.35% agar (top agar) medium in 6 well-plates coated with 0.7% agar (based agar) for 2 weeks. Total colony number and colony diameter were measured using Operetta and Harmony 3.5.2 software (below).
Figure 2
Figure 2. Identification of modulators of YAP localization
A. High-content screening evaluating YAP localization. A kinase inhibitor's library was administrated to PK9 cells using a high-throughput approach (1μM, 24H). The ratio between nuclear and cytoplasmic regions was calculated and normalized to untreated controls. The Z-score was reported in a graph, positive and negative values indicate nuclear accumulation and cytoplasmic localization, respectively. Fixed cells were incubated with antibody against YAP and DAPI staining. Sub-cellular localization of endogenous YAP protein was detected by Operetta and analyzed with Harmony 3.5.2 software. B. Modulation of TEA reporter by hit compounds. PK9 cells were transiently co-transfected with pEGFPN1 or YAP, TEA reporter (8xGTIIC-Luc reporter), and Renilla luciferase to record YAP/TAZ-dependent transcriptional activity. Cells were then treated with different compounds for 24 H and the firefly luciferase signals were normalized to the ones of Renilla luciferase. Data are globally normalized to MOCK and are presented as mean±SD. C. Modulation of CTGF and CYR61 by hit compounds. The panel represent qRT-PCR for YAP/TAZ target genes CTGF and Cyr61, relative to GAPDH expression. PK9 cells were treated with 5μM of different compounds and data, normalized to MOCK, are presented as mean±SD. (*p<0.05 and **p<0.01). D. BIS I induces YAP nuclear accumulation. Western blot analysis of nuclear fraction and cytosolic fraction of YAP in PK9 and PANC1 cell lines after treatment with 1μM and 10μM of BIS I for 24H. The relative intensities of the bands are also shown (right). Data are normalized to MOCK and presented as mean±SD. (*p<0.05 and **p<0.01). E. BIS I induces YAP post-translational modifications. Filters were blotted with antibodies against YAP and YAP Ser127-P. As negative control for phosphorylation the treated sample was incubated with calf intestinal alkaline phosphatase (CIAP). As positive control for phosphorylation at Ser127, cell lysates from high density culture was used. F. BIS I does not affect the Hippo pathway. Western blot analysis of an upstream regulator of YAP, LATS1 and its phosphorylated form (Ser909). PK9 cells were treated with BIS I 10μM for 24 H. Phosphorylation of YAP and LATS1 was measured by western blot. The relative intensities of the bands were normalized to β-actin levels (right).
Figure 3
Figure 3. BIS I treatment phenocopies YAP functional ablation
A. BIS I modulates TEA reporter in YAP-dependent manner. Left panel: BIS I activates TEA reporter activity. PK9 and PANC1 cells were transiently co-transfected with YAP (O/E YAP) or without YAP (pEGFPN1) and TEA reporter (8xGTIIC-Luc reporter), then treated with BIS I 5μM for 24H. Right panel: YAP is required for TEA reporter activation. Stably YAP silenced PANC1 cells were co-transfected with TEA reporter (8xGTIIC-Luc reporter) or its empty vector (pGL4), and Renilla. The firefly luciferase signals were normalized to the ones of Renilla. (mean±SD from biological triplicates) (*p<0.05 and **p<0.01 versus MOCK of PK9 and #p<0.05 and ##p<0.01 versus MOCK of PANC1). B. BIS I modulates YAP target genes. PK9 (left panel) and PANC1 (right panel) cells were treated with 5μM BIS I for 24H. Quantitative RT-PCRs of CTGF, Cyr61, BIRC5, AREG, YAP/TAZ target genes and YAP relative to GAPDH expression with respect to MOCK are presented as mean±SD. (*p<0.05 and **p<0.01 versus MOCK). C. BIS I displaces YAP from CTGF promoter. Map of CTGF promoter region with positions of the two primers used for ChIP analysis. TSS indicates the transcription start site, while TRE indicate the previously identified TEAD responsive elements. Chromatin immunoprecipitation (ChIP) at CTGF promoter was performed using antibodies against YAP, TEF-1 and IgG as negative control. After DNA extraction and qRT-PCR, results were normalized to non immunoprecipitated sample (INPUT) and compared to IgG for statistical significance. BIS I was able to reduce the DNA enrichment observed for YAP, whereas it was ineffective against TEF-1 DNA-binding protein.
Figure 4
Figure 4. The CTGF expression level was modulated by the TGF-β and Hippo pathways in PDAC
A. BIS I inhibits TGF-β induced CTGF expression and reduces SMAD2/3 gene expression levels. PK9 cells were stably transduced with a lentiviral vector encoding shRNA targeting YAP (shYAP) or a non-targeting control shRNA (SCR). They were then treated with BIS I 5μM in the presence and absence of TGF-β 50ng/ml for 24H. The expression levels of CTGF, SMAD2 and SMAD3 were then evaluated. (*p<0.05 and ***p<0.001 versus MOCK). B. BIS I down-regulated Smad2/3 protein levels. PK9 cells were seeded and treated with BIS I 10μM for 24H. The endogenous protein level of Smad2/3 was evaluated by western blotting against Smad2/3 antibody. The relative intensities of the bands normalized by β-actin are shown below. (***p<0.001 versus MOCK). C. BIS I inhibits anchorage-independent growth of PDAC. PDAC (1.5×104 cells) were seeded on 0.35% agar (top agar) culture medium in 6 well-plated coated with 0.7% agar (based agar). Cells were treated with BIS I 10μM for 2 weeks. Total colony number and colony diameter were measured using Operetta instrument. D. The CTGF expression level was modulated by PKCδ and GSK3β in PK9 cells. PK9 cells were incubated with siRNA targeting PKCδ, GSK3β, and non-targeting control (SCR) for 72H. Quantitative RT-PCRs of CTGF relative to GAPDH expression with respect to SCR are presented as mean+SD. (*p<0.05 and **p<0.01 versus SCR). E. Genetic ablation of GSK3β suppresses SMAD expression levels in PK9. PK9 cells were incubated with siRNA targeting GSK3β, and non-targeting control (SCR) for 72H. Quantitative RT-PCRs of CTGF relative to GAPDH expression with respect to SCR are presented as mean+SD. (***p<0.01 versus SCR).
Figure 5
Figure 5. BIS I activates β-catenin and downregulates the expression level of cancer staminality genes
A. BIS I activates β-catenin signaling pathway. β-catenin induced activation of TOP-flash (TCF/LEF) luciferase reporter was performed in HEK293T and PDAC cells lines. Cells were co-transfected with TOP-flash luciferase reporter and in the presence and absence of β-catenin. BIS I and Go6976 5μM were used for 24H treatment after transfection. Data are presented as average fold induction relative to MOCK. (*p<0.05 and **p<0.01). B. BIS I modulates β-catenin nuclear localization. Western blot analysis of nuclear fraction and cytosolic fraction of β-catenin in PK9 cells after treatment with BIS-I and Go6976 10μM for 24H. The relative intensities of the bands are shown below. The relative intensities of the bands was normalized by β-actin and lamin A/C for cytosolic and nuclear protein levels, respectively. C. β-catenin regulates CD133 expression. PK9 cells were transiently transfected with indicated β-catenin plasmid for 24H. Quantitative RT-PCRs of CD133 and OCT4 relative to GAPDH expression with respect to MOCK are presented as mean±SD. (*p<0.05 and **p<0.01). D. BIS I inhibits expression of staminality markers in PK9. PK9 cells were treated with BIS I 5μM for 24H and expression levels of ABCG2, CD133, MUL1, OCT4, PAX6, and SOX2 were analyzed by qRT-PCR relative to GAPDH expression. Data are presented as mean±SD. (**p<0.01 versus MOCK). E. Genetic ablation of YAP down-regulates CD133 expression. Stemness markers were measured by qRT-PCR in stably YAP silenced PK9 cells. Data are presented as mean±SD. (**p<0.01 versus SCR). F. Genetic ablation of GSK3β down-regulates CD133 expression. PK9 cells were incubated with siRNA targeting GSK3β, and non-targeting control (SCR) for 72H. Quantitative RT-PCRs of CD133 relative to GAPDH expression with respect to SCR are presented as mean±SD. (***p<0.01 versus SCR). G. BIS-I treatment reverts the CD133 up-regulation induced by YAP overexpression. PK9 cells were transiently co-transfected with indicated plasmids (YAP and pEGFPN1) and in the presence and absence of BIS I treatment for 24H. CD133 expression was measured by qRT-PCR. Data are presented as mean±SD. (*p<0.05 and **p<0.01 versus MOCK).
Figure 6
Figure 6. BIS I reverts YAP-induced EMT in PDAC cell lines
A. Endogenous protein level of E-cadherin in PDAC cell lines. Western blot analysis of endogenous level of E-cadherin in PDAC cell lines. B. Expression level of E-cadherin mRNA in PDAC cell lines. qRT-PCR analysis of CDH1 mRNA expression was performed in PDAC cell lines. C. Both genetic ablation of YAP and BIS I treatment induce E-cadherin expression levels. SCR or stably YAP-silenced PK9 and PANC1 cells were treated with BIS I 5μM for 24H. Western blot analysis of endogenous level of YAP and E-cadherin was performed. The relative levels of endogenous E-cadherin protein (left) and mRNA levels from these lysates (right) were evaluated by immunoblot and qRT-PCR, as shown below.
Figure 7
Figure 7. Genetic ablation of YAP and BIS I treatment regulate cell migration
A. BIS I reduces cell migration synergizing with YAP silencing. Scratch assay was performed in SCR or stably YAP-silenced PANC1 cells. Images of invaded cells at 0, 24, and 48 H after scratching and treatment with BIS I were taken from a time-lapse sequence of PANC1 cell migration; wounds with consistent shape within each well were generated using 200 μl tip. Percentage of invaded cells at different time point is indicated (right panels) as calculated by ImageJ softwre. (*p<0.05 and **p<0.01). B. BIS I down-regulates VIM and ZEB1 mRNAs. PK9 cells were treated with BIS I for 24H. Expression level of VIM and ZEB1 were measured by qRT-PCR. Data are presented as mean±SD. (**p<0.01 versus MOCK). C. BIS I inhibits TFG-β induced ZEB1 expression. PK9 cells were stably transduced with a lentiviral vector encoding shRNA targeting YAP (shYAP) or a non-targeting control shRNA (SCR). They were then treated with BIS I 5μM in the presence and absence of TGF-β 50ng/ml for 24H. The expression levels of ZEB1 was evaluated. (*p<0.05, **p<0.01 and ***p<0.001). D. Overexpression of YAP reverts the effect of BIS-I on the expression of CTGF, CDH1 and VIM. PK9 cells were transiently co-transfected with YAP and pEGFPN1 plasmids and treated with BIS I for 24H. CTGF, CDH1, VIM, and ZEB1 expression levels were measured by qRT-PCR. Data are presented as mean±SD. (*p<0.05 and **p<0.01).

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