Histone Acetylation-Mediated Regulation of the Hippo Pathway

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. © 2013 Basu et al

Role of the Beta Catenin Destruction Complex in Mediating Chemotherapy-Induced Senescence-Associated Secretory Phenotype

<div><p>Cellular senescence is considered as a tumor suppressive mechanism. Recent evidence indicates however that senescent cells secrete various growth factors and cytokines, some of which may paradoxically promote cancer progression. This phenomenon termed senescence-associated secretory phenotype (SASP) must be inhibited in order for anti-proliferative agents to be effective. The present study was designed to determine whether the β-catenin destruction complex (BCDC), known to integrate the action of various growth factors and cytokines, would represent a suitable target to inhibit the activity of SASP components. For this, we carried out experiments to determine the effect of drug-induced senescence on secretion of SASP, β-catenin transactivation, and the relationship between these processes. Moreover, genetic and pharmacological approaches were used to define the implication of BCDC in mediating the effects of SASP components on cell migration and resistance to drugs. The findings indicate that drug-induced senescence was associated with expression of various Wnt ligands in addition to previously known SASP components. Beta catenin transactivation and expression of genes implicated in epithelial-mesenchymal transition (EMT) also increased in response to drug-induced SASP. These effects were prevented by Pyrvinium, a recently described activator of BCDC. Pyrvinium also suppressed the effects of SASP on cell migration and resistance to doxorubicin. Together, these findings provide insights on the potential role of BCDC in mediating the effects of drug-induced SASP on cancer cell invasion and resistance to therapy, and suggest that targeting this pathway may represent an effective approach to enhance the activity of current and prospective anti-cancer therapeutics.</p> </div

Respective roles of DNA damage and chromatin modification in regulation of the Hippo pathway.

<p><u>Panel A</u>. 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)). <u>Panel B.</u> 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). <u>Panel C.</u> Western blots depicting the effect of Belinostat on expression and/or phosphorylation of various components of the Hippo pathway in SW480 cells. <u>Panel D.</u> Expression of TAZ in MCF7 and WM 266 cells in response to Belinostat. <u>Panel E.</u> Representative data showing the effect of siRNA mediated Knockdown of HDAC1 on expression of TAZ in WM266 cells measured by Western blot.</p

Targeting BCDC inhibits β-catenin transactivation, EMT and cell migration.

<p><u>Panel A. </u>Effect of direct and indirect activators of the BCDC on β-catenin transactivation as measured by STF activity. STF transfected cells were pre-treated with the indicated effectors for 2 hrs and then exposed to CM from those pre-treated with senescence-inducing doxorubicin concentration for an additional 24 h, followed by measure of STF activity. LY: PI3 kinase inhibitor. (LY294002), PD: MAP kinase inhibitor (PD98059), Y27: Rho kinase inhibitor (Y27632), RAP: mTOR inhibitor (rapamycin). XAV: Tankyrase inhibitor (XAV939), and PYR: casein kinase 1 activator (Pyrvinium). <u>Panel B</u>. Effect of Pyrvinium (PY) on Wnt3a- mediated activation of TopFlash. Cells were exposed for 24 hours to Wnt 3a at 10 or 50 ng/ml (Wnt-10 and Wnt-50 respectively), in the absence or the presence of pyrvinium (PYR at 500 nM). Data in panels A and B represents average of three determinations ±SE. Statistical significance is shown in panel A for drug-treated cells versus control, and in panel B between Wnt-50+PYR compared to Wnt-50-treated cells (*p<0.05, **p<0.001). <u>Panels C and D</u>. Effect of cellular exposure to PYR for 24 h on the expression of Zeb1 at the protein (Western blot) and the mRNA level (RT-PCR). <u>Panel E.</u> Effect of PYR (500 nM) on cell migration as determined by the monolayer scratch assay described in the method section.</p

Role of GSK3-associated β-catenin degradation complex in mediating β-catenin transactivation by SASP.

<p><u>Panel A.</u> Effect of enhanced GSK 3β activity on β-catenin integrity. 293 cells were transfected with a constitutively active form of GSK 3β (GSK3-S9), and expression levels of this enzyme and as well as β-catenin determined by western blot. β-actin is used here as a loading control. <u>Panel B.</u> RT-PCR analysis showing expression levels of GSK 3β, β-catenin and GAPDH in transfected and non-transfected cells as described in panel A. <u>Panel C.</u> Effect of GSK 3β activation on SASP induced β-catenin transactivation. GSK3-S9 transfected and non-transfected cells were incubated in the absence or the presence of CM from those treated with senescence inducing concentration of doxorubicin. STF activity was measured after 24 h. The data represents the average of three determinations ±SE (**p<0.001).</p

Wnt signaling activation and expression of EMT markers in association with SASP.

<p><u>Panel A.</u> β-catenin transactivation, measured by the Super-Topflash luciferase reporter system (STF) in three melanoma cell lines exposed or not to increased concentrations of doxorubicin. The FopFlash construct containing mutated TCF binding sites was used to define the specificity of doxorubicin’s action. <u>Panel B</u>. Effect of conditioned media from cells pre-treated with increasing concentrations of doxorubicin on TopFlash and FopFlash activities in naïve cells (not previously exposed to the drug). <u>Panel C.</u> Effect of individual SASP components on β-catenin transactivation determined by measure of STF activity. Data in panels A, B and C represents the average of three determinations ±SE. Statistical significance is shown for drug-treated cells versus control (*p<0.05, **p<0.001). <u>Panel D.</u> Western blots showing expression of EMT genes in WM 115 cells incubated with conditioned medium from their senescent counterparts. β-actin is used as a loading control. β-cat (c): β catenin in the cytoplasm, β-cat (N): β-catenin in the nucleus.</p

Role of secreted growth factors and cytokines in mediating Belinostat-induced activation of the Hippo pathway.

<p><u>Panel A.</u> 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. <u>Panel B.</u> 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. <u>Panel C.</u> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062478#s2" target="_blank">Methods</a> 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).</p