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. 2013 May 6;8(5):e62478.
doi: 10.1371/journal.pone.0062478. Print 2013.

Histone acetylation-mediated regulation of the Hippo pathway

Affiliations

Histone acetylation-mediated regulation of the Hippo pathway

Dipanjan Basu et al. PLoS One. .

Erratum in

Abstract

The Hippo pathway is a signaling cascade recently found to play a key role in tumorigenesis therefore understanding the mechanisms that regulate it should open new opportunities for cancer treatment. Available data indicate that this pathway is controlled by signals from cell-cell junctions however the potential role of nuclear regulation has not yet been described. Here we set out to verify this possibility and define putative mechanism(s) by which it might occur. By using a luciferase reporter of the Hippo pathway, we measured the effects of different nuclear targeting drugs and found that chromatin-modifying agents, and to a lesser extent certain DNA damaging drugs, strongly induced activity of the reporter. This effect was not mediated by upstream core components (i.e. Mst, Lats) of the Hippo pathway, but through enhanced levels of the Hippo transducer TAZ. Investigation of the underlying mechanism led to the finding that cancer cell exposure to histone deacetylase inhibitors induced secretion of growth factors and cytokines, which in turn activate Akt and inhibit the GSK3 beta associated protein degradation complex in drug-affected as well as in their neighboring cells. Consequently, expression of EMT genes, cell migration and resistance to therapy were induced. These processes were suppressed by using pyrvinium, a recently described small molecule activator of the GSK 3 beta associated degradation complex. Overall, these findings shed light on a previously unrecognized phenomenon by which certain anti-cancer agents may paradoxically promote tumor progression by facilitating stabilization of the Hippo transducer TAZ and inducing cancer cell migration and resistance to therapy. Pharmacological targeting of the GSK3 beta associated degradation complex may thus represent a unique approach to treat cancer.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Respective roles of DNA damage and chromatin modification in regulation of the Hippo pathway.
Panel A. Hippo reporter activity in response to drugs tested at concentrations that induce 50% inhibition of proliferation in SW480 cells (indicated at the top of each bar). Ctl: control., Cisp: cisplatin., Dox: doxorubicin., Bel: Belinostat., TSA: Trichostatin A., AZA: 5 Azacitidine(decitabine)). Panel B. Western blot showing the effect of Belinostat on acetylation of Histone H3 at Lysine 9 (H3K9) (Upper level), and activity of Hippo reporter in MCF7 and WM 266 melanoma cells. Each bar in Panels A and B represents the average of three determinations ±SE. Statistical significance is shown for drug-treated cells compared to the corresponding untreated controls (*p<0.05, **p<0.001). Panel C. Western blots depicting the effect of Belinostat on expression and/or phosphorylation of various components of the Hippo pathway in SW480 cells. Panel D. Expression of TAZ in MCF7 and WM 266 cells in response to Belinostat. Panel E. Representative data showing the effect of siRNA mediated Knockdown of HDAC1 on expression of TAZ in WM266 cells measured by Western blot.
Figure 2
Figure 2. Regulation of Hippo downstream genes by Belinostat and role of TAZ in mediating these effects.
Panel A. Expression of TAZ target genes CTGF and Cyr61 measured by Q-PCR in the absence or the presence of Belinostat at the indicated concentrations (µM). Panel B. Representative Western blots showing the expression of EMT genes in response to Belinostat in SW480 cells (Ecad: E Cadherin, N-Cad: N cadherin). Panel C. Effect of TAZ gene overexpression on activity of the Hippo reporter. SW480 cells were transfected with the TAZ DNA construct at the indicated concentrations and activity of luciferase reporter measured after 24 hrs. Panel D. Effect of TAZ overexpression on expression of its downstream target genes. Cells were transfected by TAZ as described in panel C and expression of CTGF and Cyr 61 and Vimentin (Vim) was measured by Q-PCR. Panel E. Representative Western blots showing expression of EMT associated genes in response to TAZ overexpression (Vim: Vimentin, N-Cad: N cadherin). Data in panels A, C and D, represent average of three determinations ±SE. Statistical significance is shown for drug-treated or TAZ-transfected cells compared to the corresponding controls (*p<0.05, **p<0.001).
Figure 3
Figure 3. Belinostat-induces stabilization rather than expression of TAZ.
Panel A. expression of TAZ in response to Belinostat (5 µM) measured quantitative PCR. Panel B. Effect of Belinostat on TAZ stabilization. SW480 cells were exposed to cycloheximide (CXH) at 10 µM concentration in the absence or the presence of Belinostat (µM). Proteins were extracted at the indicated times after addition of the compounds and TAZ protein levels determined by Western blot. Band densities were quantified by the Image J software (NIH) and graphed. Data in graphs A and B represent average of three determinations ±SE. Significance (p<001) is shown in graph B between Belinostat-treated cells for 6 hours and the corresponding non-treated cells. Panels C and D. SW480 cells were transfected with genes coding for CK1ε or constitutively active GSK3 beta (GSK-S9) and TAZ protein levels determined by Western blot after 48 hours. Panel E. Effect of Belinostat on phosphorylation of Akt and GSK3 beta. The cells were exposed to the drug for 1hour in serum free medium and protein phosphorylation detected by Western blot using specific antibodies. Beta actin is used as a loading control in panels B, C, D and E.
Figure 4
Figure 4. Potential role of G-protein coupled receptors in mediating Belinostat induced activation of the Hippo pathway.
Panel A. Effect of conditioned medium from Belinostat pre-exposed cells on activation of the Hippo reporter in naïve cells (not previously exposed to the drug). SW480 cells were incubated with Belinostat at the indicated concentrations for 24 hours, the drug was then washed extensively and the cells incubated with fresh media for 24 hrs. The resulting conditioned media (CM) were then incubated for 24 hrs with naïve cells transfected with the Hippo luciferase reporter, after what the activity of the enzyme was determined. The data represent average of three determinations ±SE. Statistical significance is shown for Bel-CM-exposed cells compared to the control (**p<0.001). Panel B. Representative Western blots showing the effect of CM from Belinostat treated cells on expression of YAP and TAZ in naïve cells. Beta actin was used as a loading control. Panel C. Effect of glucagon (Gln), a GPCR antagonist, on Bel-CM induced activation of the Hippo reporter. SW480 cells transfected with the luciferase reporter were incubated in the absence or the presence of CM from cells pre-exposed to 0.5 µM Belinostat (Bel-CM), and in the absence or the presence of glucagon at the indicated concentrations. Luciferase activity was measured after 24 hrs of incubation. The data represent average of three determinations ±SE. For every Gln concentration, values were compared between Bel-CM exposed cells and those exposed to control CM (**p<0.001). Panel D. Effect of Glucagon on Belinostat-mediated increase in TAZ levels determined by western blot in cells exposed or not to Bel-CM and in the absence or presence of Gln at 5 µM. Staining with beta actin represents a loading control.
Figure 5
Figure 5. Role of secreted growth factors and cytokines in mediating Belinostat-induced activation of the Hippo pathway.
Panel A. SW480 cells were incubated with the indicated concentrations of Belinostat for 24 hours and expression levels of selected secreted factors were determined by QPCR and compared to those in control non-treated cells. Panel B. Effect of individual growth factors and cytokines on TAZ levels and phosphorylation of GSK 3 beta. Cells were incubated with the indicated soluble factors (at 100 ng/ml each) for 24 hours and proteins extracted and processed for western blot using specific antibodies to TAZ and phosphorylated GSK3 beta. Panel C. Effect of individual growth factors and cytokines on activity of the Hippo reporter. Cells transfected with the reporter construct were incubated with the indicated factors for 24 hours and luciferase activity measured as described in the Methods section. Each bar in Panels A and C represents the average of three determinations ±SE. Statistical significance is shown for treated cells compared to the corresponding untreated controls (*p<0.05, **p<0.001).
Figure 6
Figure 6. Targeting the GSK 3 beta associated destruction complex reduces TAZ levels, cancer cell migration and resistance to therapy.
Panel A. Naïve SW480 cells were exposed to conditioned medium from Belinostat (1 µM) treated counterparts (Bel-CM), in the absence or the presence of Pyrvinium (PYR) at 0.5 µM. After 24 hours, the cells were processed for Western blot with antibodies to TAZ, Vimentin (Vim) and beta actin. Panel B. Monolayer scratch assay depicting the effect of Bel-CM on cell migration and its delay by pyrvinium. MCF cells cultured until confluency and scratches introduces in the monolayer using a pipette tip. The cells were then incubated in the presence or absence of Bel-CM, with or without pyrvinium (0.5 µM) for the indicated times, representative photographs are shown. Panel C. Effect of Bel-CM and pyrvinium of cellular response to doxorubicin. SW480 cells were incubated with doxorubicin at the indicated concentration in absence or presence of Bel-CM, Pyrvinium (PYR) or both. Cell viability was determined by MTT assay as described in the Methods section and the data represented as per cent of control non-treated cells. The data represent average of three determinations ±SE. Statistical significance is shown for Bel-CM exposed cells in the absence or the presence of PYR (**p<0.001). Panel D. Effect of PYR on cellular response to other drugs. Cells were exposed to the indicated drugs in the absence or presence of Bel-CM (BCM) and pyrvinium (P). Cell viability was determined by MTT assay after 72 hours in culture. The data represent average of three determinations ±SE. Statistical significance is shown for Bel-CM exposed cells in the absence or the presence of PYR for each drug tested (*p<0.05, **p<0.001).
Figure 7
Figure 7. Schematic model depicting nuclear regulation of the Hippo pathway in drug-affected and neighboring cells.
Exposure to certain drugs affecting DNA or chromatin results in enhanced expression of secreted growth factors and cytokines. These secreted factors (SFs) may in turn signal in autocrine/paracrine manner for activation of Akt and inhibition of GSK3 beta associated degradation complex (GSK3β/DC) resulting in stabilization of TAZ oncogene. Consequently, expression of EMT genes is enhanced, leading to increased cell migration and drug resistance (DR) in both drug affected and neighboring cells. These processes can be overcome by using pyrvinium (PYR), a pharmacological activator of GSK3 beta associated degradation complex.

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