Peroxisome Proliferator ^ Activated Receptor ...

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Maja Grabacka,1,2 Przemyslaw M. Plonka,2 Krystyna Urbanska,2 and Krzysztof Reiss1. Abstract ...... Meyer GE, Shelden E, Kim B, Feldman EL. Insulin-.
Human Cancer Biology

Peroxisome Proliferator ^ Activated Receptor A Activation Decreases Metastatic Potential of Melanoma Cells In vitro via Down-Regulation of Akt Maja Grabacka,1,2 Przemyslaw M. Plonka,2 Krystyna Urbanska,2 and Krzysztof Reiss1

Abstract

Purpose: Peroxisome proliferator-activated receptors (PPAR) regulate lipid and glucose metabolism but their anticancer properties have been recently studied as well.We previously reported the antimetastatic activity of the PPARa ligand, fenofibrate, against melanoma tumors in vivo. Here we investigated possible molecular mechanisms of fenofibrate anti metastatic action. Experimental Design: Monolayer cultures of mouse (B16F10) and human (SkMell88) melanoma cell lines, soft agar assay, and cell migration assay were used in this study. In addition, we analyzed PPARa expression and its transcriptional activity in response to fenotibrate by using Western blots and liciferase-based reporter system. Results: Fenofibrate inhibited migration of B16F10 and SkMel188 cells in Transwell chambers and colony formation in soft agar.These effects were reversed by PPAR inhibitor, GW9662. Western blot analysis revealed time-dependent down-regulation of Akt and extracellular signal ^regulated kinase l/2 phosphorylation in fenofibrate-treated cells. A B16F10 cell line stably expressing constitutively active Akt mutant was resistant to fenofibrate. In contrast, Akt gene silencing with siRNA mimicked the fenofibrate action and reduced the migratory ability of B16F1O cells. In addition, fenofibrate strongly sensitized BI6FIO cells to the proapoptotic drug staurosporine, further supporting the possibility that fenofibrate-induced down-regulation of Akt function contributes to fenofibrate-mediated inhibition of metastatic potential in this experimental model. Conclusions: Our results show that the PPAR-dependent antimetastatic activity of fenofibrate involves down-regulation of Akt phosphorylation and suggest that supplementation with this drug may improve the effectiveness of melanoma chemotherapy.

The significant need to develop new therapeutic and preventive approaches for melanoma treatment focuses attention of health service and basic science all over the world. Malignant melanoma incidence has increased dramatically in recent years (1 – 3) whereas the mortality rate in melanoma patients with the vertical tumor growth phase is very high due to tumor cell penetration in the surrounding tissues, intravasation into blood or lymphatic vessels, and rapid formation of metastases. Only very thin melanoma lesions (50 Am were counted under a light microscope. Clonogenic assay. Cells were plated in six-well plates (1,000 per well) in regular culture medium with fenofibrate (25 Amol/L), staurosporine (10 nmol/L), or both. After 1 week, cells were washed with PBS, fixed, and stained in crystal violet/carbol/25% methanol (1:1:2) mixture for 20 minutes. Colonies with diameter >0.5 mm were counted. Statistical analysis. Statistical significance of the differences between groups was tested using the Student’s t test for homogeneous or heterogeneous variances, as appropriate, or one-way ANOVA (significance level, 5%).

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Human Cancer Biology

Results Active PPARa is expressed in B16F10 melanoma cells. We previously reported the detection of PPARa in hamster melanoma cell lines and the attenuation of metastatic spread of these cells by a synthetic PPARa agonist, fenofibrate (22). To investigate the cellular and molecular mechanisms underlying the action of fenofibrate in melanoma, we used a highly metastatic murine melanoma cell line, B16F10 (23, 24). The results in Fig. 1A show that B16F10 cells express PPARa protein at a level comparable to the control brown adipose tissue and much lower than in liver. The evaluation of PPARa transcriptional activity in B16F10 cells showed >2-fold stimulation of PPAR responsive elements by fenofibrate (Fig. 1B), further confirming the presence of an active PPARa signaling system in these malignant cells. Fenofibrate decreases migration and anchorage-independent growth of mouse and human melanoma cell lines. The ability to migrate, cell survival, and anchorage-independent proliferation are the hallmarks of malignant transformation. To determine whether PPARa activation affects those properties of B16F10 cells, we evaluated cell migration in Transwell chambers and colony formation in soft agar after the treatment with fenofibrate. As shown in Fig. 2A and C, fenofibrate remarkably decreased the migration of B16F10 and SkMel cells. Reductions of >4-fold and >2-fold in the number of migratory B16F10 and SkMel cells, respectively, were observed in the presence of 25 Amol/L fenofibrate. In B16F10 cells, migration in the presence of fenofibrate and GW9662, a synthetic PPARg inhibitor, was tested. In higher concentration (10 Amol/L), GW9662 also blocks PPARa (28) and efficiently restores cell migration, indicating that fenofibrate action on cell migration is PPARa dependent. Because cell proliferation could affect cell counts in

Fig. 1. PPARa is present and active in B16F10 melanoma cells. A, expression of PPARa in B16F10 cells. Liver and brown adipose tissue (BAT) extracts serve as positive controls. The membrane was stripped and probed for GRB2 to show equal gel loading. B, fenofibrate (F ; 25 Amol/L) induces transcriptional activity of PPARa. Reporter plasmid contains luciferase gene driven by PPAR responsive element (PPRE) consisting of three copies of J site from apo-AII gene promoter (25, 26). Columns, mean from four independent experiments (n = 11); bars, SE. *, P < 0.0001.

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the migration assay, we evaluated the contribution of this variable in our experimental setting. As shown in Fig. 2A (right), the increase in cell number was minimal in the condition in which cell migration was assessed (SFM and SFM + F), providing further confirmation that fenofibrate inhibited the ability of B16 cells to cross through the pores of the polycarbonate membrane. Importantly, fenofibrate partially inhibited also colony formation in soft agar. Results in Fig. 2B and D show 50% and 75% inhibition of colony formation in the presence of 25 Amol/L fenofibrate for B16F10 and SkMel cells, respectively. This statistically significant inhibition in B16F10 was restored when the fenofibrate treatment was accompanied by the PPAR inhibitor GW9662. In conclusion, fenofibrate treatment leads to impaired cell migration and the attenuation of anchorage-independent growth. The next experiments were designed to test whether alterations in specific cell signaling pathways could account for the changes in cellular behavior after fenofibrate treatment. Fenofibrate-activated PPARa interferes with Akt and Erk1/2, but not GSK3b, signaling cascades. In the next step, we evaluated the phosphorylation status of Akt, Erk1/2, and GSK3h, three signaling molecules known to play a role in malignant transformation. We observed a time-dependent down-regulation of Ser473 phoshorylation of Akt after incubation with fenofibrate (25 Amol/L) without a change in total Akt protein level (Fig. 3A). B16F10 cells possess a high basal level of Akt phosphorylation (control), which was not affected by DMSO used as a vehicle. A similar down-regulation in phosphorylation after fenofibrate treatment was noted for Erk1/2, but not for GSK3h. Quantitatively, fenofibrate induced >3-fold decrease in Akt phosphorylation and 24-fold decrease in Erk1/2 phosphorylation from the basal level over the 48-hour incubation time. To prove that these effects were PPARa dependent, we treated the cells with fenofibrate (25 Amol/L) together with the PPAR inhibitor GW9662 (10 Amol/L) and compared the Akt and Erk1/2 phosphorylation status at corresponding time points. As shown in Fig. 3B, PPARa inhibition totally abolished the action of fenofibrate. These results show that activated PPARa can interfere with both Akt and Erk1/2 signaling cascades. The next set of experiments was designed to determine whether impairment of signal transduction via Akt or Erk1/2 is responsible for fenofibrate-induced inhibition of melanoma cell migration and growth. The decrease of B16F10 migration and anchorage-independent growth by fenofibrate is Akt dependent but not Erk1/2 dependent. The results depicted in Fig. 4A show that Akt is continuously phoshorylated when B16F10 cells are incubated in SFM. To further assess the involvement of Akt in PPAR-mediated inhibition of B16F10 migration and anchorage-independent growth, we developed a stable B16F10 cell line expressing constitutively activated (myristoylated) Akt (B16/myrAkt). In such cells, the level Akt is several times higher than in parental cells and its phosphorylation is strongly promoted by constitutive membrane association of the myristoylated protein (29). The migration assay of B16/myrAkt cells showed that fenofibrate was no longer able to inhibit either migration or colony formation in soft agar (Fig. 4B and C, respectively) when the constitutively active Akt was expressed. Importantly, the inhibitory effect of fenofibrate on cell migration was efficiently imitated by silencing the Akt

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Active PPARa Inhibits Melanoma Growth and Migration

Fig. 2. Fenofibrate decreases cell migration and anchorage-independent growth of melanoma B16F10 and SkMel 188. A, fenofibrate inhibits cell migration inTranswell chambers (left). Cells (5  104) were suspended in 200 AL of SFM with 25 Amol/L fenofibrate (F) or DMSO (Control) and seeded in the upper chamber; after 48 hours, the inserts were washed in PBS and wiped with cotton swabs to remove the cells which did not migrate. The cells on the bottom surface of the filters were stained with crystal violet and counted under a microscope. GW8662 (10 Amol/L), a PPAR inhibitor, restores the migratory ability in fenofibrate-treated cells. Columns, mean from five independent experiments (n = 17, n = 16) for F and Control group or from two experiments (n = 7, n = 8) for GW9662 and F + GW9662 group, respectively; bars, SE. **, P < 0.0001; *, P < 0.05, between control and F and between F and GW9662 + F, respectively. Serum deprivation decreases B16F10 cell proliferation (right). Cells were seeded in a 24-well plate at a density of 5  103 per well and maintained in serum-containing medium (10% FBS), SFM alone, or supplemented with 25 Amol/L fenofibrate (SFM + F). At indicated time points, cells were harvested, stained with trypan blue, and counted under a microscope. Columns, mean (n = 4); bars, SD. B, fenofibrate inhibits colony formation in soft agar. Cells (1  104) suspended in 0.4% agarose/DMEM with 10% FBS and fenofibrate or DMSO were plated onto 0.8% agarose/DMEM with 10% FBS. After 10 days, colonies >50 Am were counted. GW9662 (10 Amol/L) significantly decreases the effect of fenofibrate. Columns, mean from five independent experiments (n = 22, n = 23) for F and Control group, or from two experiments (n = 13, n = 18) for GW9662 and F + GW9662 group, respectively; bars, SE. **, P < 0.0001; *, P < 0.0001, between control and F and between F and GW9662 + F, respectively. C, fenofibrate inhibits SkMel 188 cells inTranswell chambers. The assay was done as for B16F10, but with lower cell density (3  104), and the migratory cells were fixed, stained, and counted after 24 hours. Columns, mean (n = 6); bars, SD. *, P < 0.001. D, fenofibrate inhibits SkMel 188 colony formation in soft agar. The conditions of the assay were the same as for B16F10 cells. Columns, mean (n = 20); bars, SD. *, P < 0.00001.

expression with specific siRNA (Fig. 4D). Irrelevant siRNA against lamin A and mock-transfected cells served as controls. These results confirm that active Akt is important for B16F10 migration, which is in agreement with other reports on melanoma invasive behavior (30). This also supports that the observed action of fenofibrate on B16F10 migration is, at least in part, Akt dependent. In contrast to Akt phosphorylation, serum starvation of B16F10 cells resulted in significant down-regulation of Erk1/2 phosphorylation (Fig. 5A). Because the migration assays were done in serum-free conditions, the involvement of Erk1/2 activity in this process was probably less pronounced. To evaluate such a possibility, we used the synthetic inhibitor of mitogen-activated protein kinase/Erk kinase, U0126, which blocks Erk1/2 phosphorylation (31). Indeed, in B16F10 cells, U0126, even in a concentration as low as 0.5 Amol/L, abolished Erk1/2 phosphorylation (Fig. 5B). U0126 at 0.5 Amol/L was therefore used in the migration and soft-agar colony formation assays of B16F10 and B16/myrAkt cells (Fig. 5C and D, respectively). In contrast to Akt, Erk1/2 inhibition did not decrease migration and colony formation of B16F10 cells. Therefore, it is reasonable to assume that although Erk1/2 phosphorylation is inhibited by fenofibrate, it does not contribute to the fenofibrate effects on migration and colony formation.

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Fenofibrate reveals synergy with staurosporine against B16F10 melanoma growth. The crucial role of Akt phosphorylation and its down-regulation by fenofibrate led us to speculate that fenofibrate could be additive to the antimelanoma activity of selected cytotoxic agents such as staurosporine. Shutdown of the phosphatidylinositol 3-kinase/Akt pathway, which usually confers a powerful prosurvival signaling cascade, could potently increase the effect of apoptosis-inducing chemotherapeutics, such as staurosporine. Staurosporine and its derivatives, which are protein kinase C inhibitors, have already been used to treat advanced metastatic melanoma in phase I and II of clinical trials (32, 33). Cell survival in monolayer cultures in Fig. 6A shows dosedependent decrease in cell proliferation for fenofibrate (left) and decrease of both cell proliferation and cell survival for staurosporine (right), evaluated 48 hours after the treatment. Fenofibrate, even in high concentrations (50 and 100 Amol/ L), did not cause cell loss, but rather blocked proliferation. Staurosporine, however, was cytotoxic in concentrations exceeding 100 nmol/L. We chose fenofibrate concentration of 25 Amol/L (used in the experiments described above) and a mildly effective staurosporine concentration of 10 nmol/L to check their potentially synergistic effect on B16F10 cell proliferation and survival. Untreated B16F10 grew exponentially in the presence of 10% FBS whereas both staurosporine

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and fenofibrate inhibited cell proliferation in these culture conditions (Fig. 6B). The most remarkable growth inhibition was noted for combined fenofibrate and staurosporine 96 hours after the treatment. In comparison with fenofibrate alone, the inhibition was 4-fold, and in comparison with staurosporine, the inhibition was >8-fold greater. Synergistic effects of low concentration of fenofibrate and staurosporine on B16F10 growth were even more spectacular when tested in clonogenic assay. Figure 6C shows complete inhibition of colony formation following single-dose treatment of B16F10 cells with 10 nmol/L staurosporine and 25 Amol/L fenofibrate.

Discussion In this article, we show that mouse (B16F10) and human (SkMel 188) melanoma cell lines express PPARa, and its activation by fenofibrate causes inhibition of migration and anchorage-independent growth. It is consistent with our previous report that fenofibrate administered orally to melanoma-bearing hamsters significantly decreases the metastatic

spread from the primary site (22). We found that fenofibrate inhibits both Erk1/2 and Akt phosphorylation in a PPARadependent fashion, although only the latter event is functionally linked with fenofibrate-mediated growth repression and inhibition of migration. Both phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase/Erk1/2 pathways play important roles in normal and malignant cell migration (34 – 37). PPARs have been reported to interfere with both these pathways. PPARg and PPARa ligands, including fenofibrate, inhibit vascular endothelial growth factor – and basic fibroblast growth factor – induced endothelial cell migration, which is accompanied by a decrease in Akt phosphorylation (38, 39). In contrast, some other authors report a rapid but transient increase of Erk1/2 and Akt phosphorylation caused by PPARa and PPARg ligands in cell culture conditions (40 – 42). However, in these studies, changes in the phosphorylation status were detected shortly (10-30 minutes) after the treatment and therefore cannot be ascribed to canonical activity of PPARs as transcription factors. Such rapid, ‘‘nongenomic’’ effects are most likely PPAR independent (42, 43). In our case, fenofibrate

Fig. 3. Active PPARa interferes with Akt and Erk1/2 signaling pathways in B16F10 cells. A, fenofibrate induces time-dependent decrease in Akt and Erk1/2 phosphorylation but does not influence GSK3h phosphorylation. The densitometric analysis of the protein bands from corresponding time points and control (NT) cells is shown in the graphs. B16F10 cells were cultured in the medium containing 10% FBS and DMSO or 25 Amol/L fenofibrate and the protein extracts were prepared at the indicated time points. B, the effect of fenofibrate on Akt and Erk1/ 2 phosphorylation is abolished by GW9662. B16F10 cells were cultured with 10 Amol/L GW9662 or 25 Amol/L fenofibrate + 10 Amol/L GW9662 (F + GW9662) for the indicated periods of time. The membranes were probed for total Akt, Erk1/2, and GRB2 to control the quality of cell lysates and to show equal gel loading.

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Active PPARa Inhibits Melanoma Growth and Migration

Fig. 4. Inhibition of B16F10 migration and anchorage-independent growth by fenofibrate requires Akt down-regulation. A, serum starvation does not affect Akt phosphorylation status, which is similar after incubation in serum-containing (10% FBS) medium or in SFM for 24 and 48 hours. B, overexpression of constitutively activated Akt abolishes fenofibrate effect on cell migration. A stable B16/myrAkt clone was treated with 25 Amol/L fenofibrate or DMSO. Columns, mean from two independent experiments (n = 6); bars, SD. C, constitutive Akt activation restores fenofibrate-induced inhibition of anchorage-independent growth. Soft-agar colony-forming assay of B16/myrAkt treated with 25 Amol/L fenofibrate or DMSO. Columns, mean from two independent experiments (n = 10); bars, SD. *, P < 0.05. D, Akt silencing by introduction of siRNA causes inhibition of B16F10 cell migration. Anti ^ lamin A siRNA and mock-transfected cells serve as controls. Columns, mean from two independent experiments (n = 6); bars, SD. *, P < 0.05. Inside panel shows decrease in Akt and lamin A protein level 48 and 72 hours after transfection. The membrane was stripped and probed for GRB2 to show equal gel loading.

induced PPARa transcriptional activity and the biological consequences are long-term and stable for several days after the initial administration. This time scale draws the attention to potential therapeutic applications and relevance to cancer treatment.

Akt, a cellular homologue of a retroviral oncoprotein v-Akt, is a kinase recruited during signal transduction from growth factor receptors and intracellular pathways. Its activity is regulated by association with the plasma membrane, where it interacts with phosphatidyl inositide phosphate residues, and

Fig. 5. Erk1/2 is not involved in PPARa-mediated inhibition of migration and anchorage-independent growth of B16F10 cells. A, phosphorylation of Erk1/2 is decreased in B16F10 cells serum starved for 24 and 48 hours. GRB2 labeling serves as a loading control. B, mitogen-activated protein kinase/Erk kinase inhibitor, U0126, abolishes Erk1/2 phosphorylation. B16F10 cells were cultured in serum-containing medium and incubated for 30 minutes with DMSO or the indicated concentrations of U0126. C, inhibition of Erk1/2 phosphorylation does not affect cell migration. B16F10 cells expressing activated Akt (B16/myrAkt) or B16F10 parental cells were incubated with 0.5 Amol/L U0126 or DMSO during the migration assay. Columns, mean (n = 4); bars, SD. D, inhibition of Erk1/2 phosphorylation has no effect on the colony-forming ability of parental B16F10 or B16/myrAkt cells. Columns, mean (n = 4); bars, SD.

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Fig. 6. Fenofibrate increases sensitivity of B16F10 cells to staurosporine. A, dose-responsive inhibition of cell proliferation after fenofibrate and staurosporine treatment. Columns, mean change in cell number as a percent of starting cell number (5,000 cells; n = 3); bars, SD. B, 25 Amol/L fenofibrate and 10 nmol/L staurosporine synergistically inhibit cell proliferation. Columns, mean change in cell number as a percent of starting cell number (5,000 cells; n = 6); bars, SD. C, clonogenic assay of B16F10 cells treated with 25 Amol/L fenofibrate, 10 nmol/L staurosporine (S), or combination of both (S + F). Fenofibrate enhances the negative effect of staurosporine on cell proliferation. Columns, mean number of colonies >0.5 mm; bars, SD. Pictures below show the colonies after 1week of culture, fixed and stained with crystal violet.

by subsequent phosphorylation on Thr308 and Ser473 by phosphoinositide-dependent kinases 1 and 2, recently identified as DNA-dependent protein kinase (44, 45). Akt has maximal kinase activity only when phosphorylated at both sites (20, 45). We have shown that overexpression of a constitutively active Akt mutant rescues cells from fenofibrate-induced inhibition of migration and anchorage-independent growth. This mutant contains the myristoylation signal from src protein, which is responsible for the membrane localization, and possesses several-fold higher activity than wild-type Akt due to hyperphosphorylation (29). Apparently, this hyperphosphorylation makes Akt resistant to fenofibrate. Most likely, the inhibitory action of PPARa on Akt does not involve a direct interaction between these two proteins but rather requires at least one of the proteins regulated by PPARa at the transcriptional level. Phosphatase and tensin homologue is known to inhibit Akt and block its recruitment to the membrane (46) but there are no reports of this protein being regulated by PPARa; indeed, we did not observe any change in phosphatase and tensin homologue level after fenofibrate treatment (not shown).

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A good candidate to play a role in PPARa-mediated Akt inhibition is TRB3, a mammalian homologue of Drosophila tribbles. TRB3 blocks insulin signaling pathway and its main physiologic function is to promote glucose output from liver and switch to fatty acid oxidation under fasting conditions (47). It does this task in tight cooperation with PPARa and PPARg coactivator-1 (48). TRB3 inhibits Akt phosphorylation on both Ser473 and Thr308 sites by direct binding to Akt, and therefore prevents its activation (47). TRB3 promoter contains numerous functional PPAR responsive element consensus sites and activation of PPARa induces transcription of TRB3 and hepatic glucose production in vivo as a result of Akt inhibition by TRB3 (48). It remains to be verified if PPARa-induced Akt inhibition in B16F10 cells is TRB3 mediated. Interestingly, interactions between Akt and PPARa in regulation of glucose metabolism are mutual because in mice with cardiac overexpression of myristoylated Akt, PPARa and PPARg coactivator-1 transcription is severely down-regulated (49). A similar situation might be in B16F10/myrAkt transfectants and it could explain why this cell line was resistant to fenofibrate.

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Active PPARa Inhibits Melanoma Growth and Migration

Akt is a key member of a signaling cascade coordinating prosurvival and antiapoptotic processes, as well as cell cycle progression (20, 50). Direct involvement of Akt in tumor cell invasion has been reported (21). Constitutive activation of Akt is observed in various tumors and is correlated with their progression and level of malignancy (50 – 52). In melanoma cells, a high basal level of Akt phosphorylation is frequently observed and may be essential for invasion and metastatic spread (30, 53). This is the case in B16F10 cells, which were selected as a highly invasive variant of the B16 murine melanoma (23, 24). Decrease of high basal level of Akt phosphorylation by fenofibrate in cancer cells can have a therapeutic application in both early and advanced stages of malignant melanoma. The great advantage of fenofibrate is its low toxicity and the fact that it is routinely used to treat hyperlipidemia and hypercholesterolemia. Besides its beneficial effects in reducing the risk of cardiovascular disease and atherosclerosis progression, fibrate drugs have been reported to lower the incidence of various types of cancer, including melanoma (8 – 10). These observations have led the authors to hypothesize that lipid-lowering drugs could decrease the risk of melanoma development and implied the potential chemopreventive role for these compounds (8, 9). In this article, we asked whether fenofibrate could enhance the effect of chemotherapeutic drugs through inhibition of Akt. We chose staurosporine, a protein kinase C inhibitor

with a strong proapoptotic activity, which is currently tested in clinical trials of patients with various tumors including metastatic melanoma (32, 33). Indeed, we observed that fenofibrate and staurosporine are much stronger inhibitors of the proliferation and clonogenic growth of B16F10 melanoma cells when administered together rather than separately. This synergistic effect can be attributed to the fact that staurosporine inhibits phosphoinositide-dependent kinase 1 and, consequently, Thr308 but not Ser473 phosphorylation of Akt (54). Fenofibrate, in parallel, down-regulates Ser473 phosphorylation (Fig. 3A) and therefore enhances the effect of staurosporine. This synergy in antitumor action between fenofibrate and staurosporine is a novel observation and could be applied to develop new therapeutic regimens for patients with metastatic disease. The fenofibrate concentration used in our in vitro experiments is comparable to plasma concentrations of fenofibric acid, an active metabolite of fenofibrate, detected in patients during standard hyperlipidemia treatment: 300 mg regular or 250 mg slow-release capsules daily. In such patients, plateau-phase plasma concentration of fenofibric acid is f10 to 12 Ag/mL (28-33 Amol/L; ref. 55). Therefore, as a systemic chemotherapy regimen, the combination of fenofibrate and staurosporine also holds a promise for tolerability and low toxicity compared with current regimens, of which many are highly toxic to the patient.

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Peroxisome Proliferator−Activated Receptor α Activation Decreases Metastatic Potential of Melanoma Cells In vitro via Down-Regulation of Akt Maja Grabacka, Przemyslaw M. Plonka, Krystyna Urbanska, et al. Clin Cancer Res 2006;12:3028-3036.

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