Journal of Dermatological Science 78 (2015) 44–50

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PDK1 is a potential therapeutic target against angiosarcoma cells Makoto Wada a,b,*, Mano Horinaka b, Shusuke Yasuda b, Mikio Masuzawa c, Toshiyuki Sakai b, Norito Katoh a a

Department of Dermatology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan Department of Molecular-Targeting Cancer Prevention, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan c Department of Regulation Biochemistry, Kitasato University School of Allied Health Sciences, Kanagawa, Japan b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 21 August 2014 Received in revised form 24 January 2015 Accepted 27 January 2015

Background: Angiosarcoma is a rare and aggressive malignant neoplasm of endothelial cells. Recent studies have shown that the mTOR pathway is also aberrantly activated in cutaneous angiosarcoma. New therapeutic strategies are required because the prognosis of this disease is still poor. Objective: The aim of the present study was to determine the driver gene of angiosarcoma useful for the screening of small molecule inhibitors. Methods: We investigated the sensitivity of inhibitors for the PI3K/AKT/mTOR pathway in ISOS-1 and ISO-HAS cutaneous angiosarcoma cell lines. Flow cytometric analysis was conducted to evaluate cellcycle progression and apoptosis. Western blot analyses were performed to elucidate the possible underlying mechanisms of growth inhibition. The colony formation assay was conducted to evaluate the clonogenic potential. We used the siRNA for PDK1 to examine the role of PDK1 on the growth of angiosarcoma cells. Results: The PI3K inhibitor and mTOR inhibitor inhibited the growth of both cell lines in a dosedependent manner. The PI3K inhibitor more effectively induced cell-cycle arrest at the G1 phase with the downregulated expression of cyclin D in ISOS-1 cells than the mTOR inhibitor. The PI3K inhibitor and mTOR inhibitor weakly but significantly induced G1 cell cycle arrest at the same degree in ISO-HAS cells. The expression of cyclin D was downregulated by the treatment with siRNA for PDK1, but not by the AKT inhibitor in ISOS-1 and ISO-HAS cells. The knock down of PDK1 with siRNA was more effective at reducing colony numbers than the mTOR inhibitor in ISOS-1 cells. Conclusion: These data showed that PDK1 played a pivotal role in the growth of angiosarcoma cells. Therefore, inhibition of PDK1, but not AKT, may be a more appropriate strategy than that of mTORC1 for the treatment of cutaneous angiosarcoma; the PDK1 inhibitor is promising as a therapeutic agent. ß 2015 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Keywords: PDK1 Cyclin D AKT-independent

1. Introduction Angiosarcoma is a subtype of soft tissue sarcoma, an aggressive malignant neoplasm of endothelial cells. Although angiosarcoma can occur in any organ, the most commonly affected organ is the skin, especially the scalps and faces of elderly people. Although to treat patients surgery, radiation, or chemotherapy using docetaxel have been aggressively used, their prognoses are still poor with the overall 5-year survival rate being approximately 35% [1]. Since

* Corresponding author at: Department of Dermatology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan. Tel.: +81 75 251 5586; fax: +81 75 251 5586. E-mail address: [email protected] (M. Wada).

angiosarcoma is considered to arise from endothelial cells, VEGF and its receptors are believed to be attractive therapeutic targets and several findings have been reported from clinical trials using the VEGF-A monoclonal antibody, bevacizumab, and multiple kinase inhibitor, sorafenib or sunitinib [2]. However, targeting agents for VEGFR only led to partial responses, and these became resistant within about 6 months. Recent molecular technologies revealed several mutations including VEGFR2, KRAS, and TP53 in angiosarcoma [3–7]. As a downstream signal of KRAS, the PI3K-AKT pathway is considered to be more important than the MAPK pathway in angiosarcoma [8,9], and rapamycin has been shown to suppress the growth of angiosarcoma cells both in vitro and in vivo [10,11]. However, crucial targets for the treatment of angiosarcoma are still insufficient. Therefore, we investigated the antiproliferative effects

http://dx.doi.org/10.1016/j.jdermsci.2015.01.015 0923-1811/ß 2015 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

M. Wada et al. / Journal of Dermatological Science 78 (2015) 44–50

of several small molecular agents that target the downstream molecules of RAS in angiosarcoma cells, and found that phosphoinositide-dependent protein kinase 1 (PDK1) was a promising target against angiosarcoma.

2. Materials and methods

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2.4. Cell-cycle analysis Cells were incubated with or without agents as indicated and then harvested. They were trypsinized and stained with 10 mg/ mL of propidium iodide. Flow cytometry was carried out using a FACScalibur (Becton Dickinson). Data were analyzed with CellQuest software (Becton Dickinson) and Modifit software (Verity Software House).

2.1. Cell culture 2.5. Western blot analysis ISOS-1 and ISO-HAS cells were obtained from the Cell Resource Center for the Biomedical Research Institute of Development, Aging and Cancer, Tohoku University. Each cell line was expanded and placed in stock within a month of receipt. ISOS-1 cells were maintained in DMEM. ISO-HAS cells were maintained in angiosarcoma cell-culture medium, as previously reported [12]. Culture media were supplemented with 10% fetal bovine serum, 4 mM L-glutamine, 50 U/mL penicillin, and 100 mg/mL streptomycin. Cell cultures were incubated at 37 8C in a humidified atmosphere of 5% CO2. 2.2. Reagents LY294002, NVP-BEZ235, everolimus (RAD001), PD0325901, PLX4720, MK2206, and OSU-03012 were purchased from Selleck Chemicals. All compounds were dissolved in dimethyl sulfoxide (DMSO) as stock and stored at 80 8C. 2.3. Cell viability assay The number of viable cells was determined using the Cell Counting Kit-8 assay according to the manufacturer’s instructions (Dojindo). After being incubated with the indicated concentrations of various agents for 48 h for ISOS-1 cells or 72 h for ISO-HAS cells, the kit reagent WST-8 was added to the medium and both cell lines were incubated for a further 4 h. The absorbance of samples (450 nm) was determined using a scanning multiwell spectrophotometer that served as an ELISA reader.

Cells were incubated with or without agents as indicated and then harvested. The cells were re-suspended in lysis buffer (50 mM Tris–HCl, 1% SDS, 2 mg/mL leupeptin, 2 mg/mL aprotinin, 0.5 mM phenylmethylsulfonyl fluoride, and 0.1% 2-mercaptoethanol). The lysate was sonicated and centrifuged at 15,000  g for 20 min at 4 8C, and the supernatant was collected. Equal amounts of lysate were analyzed by SDS-PAGE and transferred to PVDF membranes (Millipore). Primary antibodies were obtained for the following proteins: AKT (no. 9272), phospho-AKT Thr308 (no. 9275), phospho-AKT Ser473 (no. 9271), cyclin D2 (no. 3741), phospho-S6K Thr389 (no. 9234), phospho-S6K Thr421/424 (no. 9204), S6K (no. 2708), phospho4E-BP1 Thr37/46 (no. 2855), phospho-4E-BP1 Ser65 (no. 9451), 4E-BP1 (no. 9644), phospho-RB Ser780 (no. 9307), phospho-RB Ser807/811 (no. 9308) (Cell Signaling Technology, Inc.), p27 (sc528), cyclinE (sc-247), cyclin D3 (sc-182), CDK2 (sc-163), CDK4 (sc-601) (Santa Cruz Biotechnology, Inc.), b-actin (Sigma), RB (clone G3-245), p21 (clone 6B6) (BD Bioscience), and cyclin D1 (clone DSC-6) (Medical and Biologic Laboratories). The blots were blocked in blocking buffer (5% skim milk/TBST) for 1 h at room temperature, and incubated with the appropriate primary antibody in blocking buffer for 1 h at room temperature. The blots were then washed and incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h, and signals were detected with the Immobilon Western Chemiluminescent HRP Substrate (Millipore).

Fig. 1. Effects of the PI3K or mTOR inhibitor on angiosarcoma cells. Cells were treated with the PI3K or mTOR inhibitor, LY294002, NVP-BEZ235, or everolimus, at various concentrations for 48 h for ISOS-1 cells or 72 h for ISO-HAS cells. Cell viabilities were determined using the WST-8 assay. Data represent the means of triplicates with SD indicated. Significance was assessed by Student’s t test. * P < 0.05; ** P < 0.01 significantly different from DMSO-treated controls. NT; no treatment.

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Fig. 2. Effects of the PI3K or mTOR inhibitor on cell-cycle progression. (A) ISOS-1 cells were treated with the indicated concentrations of the PI3K or mTOR inhibitor for 12 h. The percentage of cells in each phase of the cell cycle was determined by flow cytometry. Data represent the means of triplicates with SD indicated. Significance was assessed by Student’s t test. * P < 0.05 significantly different from DMSO-treated controls. (B) Representative histogram patterns from ISOS-1 cells. (C) ISO-HAS cells were treated with the indicated concentrations of the PI3K or mTOR inhibitor for 72 h. The percentage of cells in each phase of the cell cycle was determined by flow cytometry. Data represent the means of triplicates with SD indicated. Significance was assessed by Student’s t test. * P < 0.05 significantly different from DMSO-treated controls. (D) Representative histogram patterns from ISO-HAS cells. (E) Cells were treated with the indicated concentrations of the PI3K or mTOR inhibitor for 12 h in ISOS-1 cells, for 72 h in ISO-HAS cells, and cell extracts were analyzed by immunoblotting. LY; LY294002, BEZ; NVP-BEZ235, EV; everolimus.

2.6. Small interfering RNA transfection StealthTM RNAi Negative Control Duplexes and Small interfering RNAs (siRNA) for PDK1 were obtained from Invitrogen,

(50 -CGAACUCCUUUGAACUGGACUUACA-30 ) for ISOS-1 cells, (50 -GAAAUGGAAGGAUACGGACCUCUUA-30 ) for ISO-HAS cells. Cells were transfected with 20 nM of each siRNA using the Invitrogen RNAi MAX reagent. Six hours after the transfection, cells

M. Wada et al. / Journal of Dermatological Science 78 (2015) 44–50

were incubated with fresh media. Cells were harvested for immunoblotting after 48 h in ISOS-1 cells, 72 h in ISO-HAS cells, or seeded for the colony formation assay. 2.7. Colony formation assay Cells were seeded at a density of 100 cells in each well of the 6-well plates. The cells were treated with each agent for 48 h. After 48 h, agent-treated cells were then washed twice in PBS followed by the addition of fresh media. After being incubated for 10 more days, cells were fixed with 10% formalin, and stained with 0.1% crystal violet in 20% ethanol. 2.8. Statistical analysis Data were expressed as the mean  SD of three determinations. Significance was assessed by Student’s t-test. A value of P < 0.05 was considered to be significantly different from DMSO-treated controls. 3. Results

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displayed apparently weaker responses to the MEK inhibitor PD0325901 at a maximum concentration of 1 mM (Supplementary Fig. S1). We next investigated the effects of the PI3K inhibitor, dual PI3K/mTOR inhibitor, and mTOR inhibitor on cell-cycle progression and apoptosis in each cell line using flow cytometric analysis. As shown in Fig. 2A and B, LY294002 and NVP-BEZ235 more effectively induced G1 cell-cycle arrest than everolimus in ISOS1 cells. On the other hand, each inhibitor weakly but significantly induced G1 cell-cycle arrest at the same degree (Fig. 2C and D), and LY294002 at the maximum concentration of 50 mM clearly induced apoptosis in ISO-HAS cells (Supplementary Fig. S2). We then investigated the mechanism underlying G1 cell-cycle arrest in both cells by Western blotting. As shown in Fig. 2E, LY294002 only decreased the expression of the phosho-AKT (Thr308) and phospho-AKT (Ser473) in both cells. LY294002, NVPBEZ235, and everolimus similarly reduced phospho-S6K. LY294002 more strongly increased the expression of p27 than NVP-BEZ235 or everolimus in ISOS-1 cells. LY294002 and NVPBEZ235 more effectively decreased cyclin D and phospho-4E-BP1 levels than everolimus in both cells. Phosphorylation of the RB protein was inhibited by each inhibitor in ISOS-1 cells.

3.1. Effects of PI3K and mTOR inhibitors on angiosarcoma cells 3.2. AKT-independent cell growth To investigate the antiproliferative effects of the PI3K and mTOR inhibitors on angiosarcoma ISOS-1 and ISO-HAS cells, we used the PI3K inhibitor LY294002, dual PI3K/mTOR inhibitor NVP-BEZ235, and mTOR inhibitor everolimus (RAD001). The IC50 of each agent was determined using the WST-8 assay. The PI3K inhibitor and dual PI3K/mTOR inhibitor inhibited growth by approximately 80%, whereas the mTOR inhibitor inhibited growth by 50–60% (Fig. 1). Each cell line was resistant to the RAF inhibitor PLX4720, and

AKT is known to be activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 and by phosphorylation within the carboxy terminus at Ser473 by mTORC2 [13–15]. AKT plays a role in regulating cell-cycle by preventing GSK-3b-mediated phosphorylation and degradation of cyclin D1 [16]. We used AKT kinase inhibitor MK2206 to investigate the AKT kinase activity in relation to the expression

Fig. 3. The AKT inhibitor MK2206 did not regulate cyclin D levels in angiosarcoma cells. (A) cells were treated with the AKT inhibitor MK2206 at the various concentrations for 48 h in ISOS-1 cells, for 72 h in ISO-HAS cells, and cell viabilities were determined using the WST-8 assay. Data represent the means of triplicates with SD indicated. (B) Cells were treated with the indicated concentrations of the AKT inhibitor for 12 h in ISOS-1 cells, for 72 h in ISO-HAS cells. Cell extracts were analyzed by immunoblotting for the indicated proteins. NT; no treatment.

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Fig. 4. PDK1-dependent cell growth. (A) Cells were treated with PDK1 inhibitor OSU-03012 at the various concentrations for 48 h in ISOS-1 cells, for 72 h in ISO-HAS cells, and cell viabilities were determined using the WST-8 assay. Data represent the means of triplicates with SD indicated. Significance was assessed by Student’s t test. * P < 0.05; ** P < 0.01 significantly different from DMSO-treated controls, NT; no treatment. (B) Cells were treated with the indicated concentrations of the AKT inhibitor for 12 h in ISOS-1 cells, for 72 h in ISO-HAS cells. Cell extracts were analyzed by immunoblotting for the indicated proteins. (C) ISOS-1 and ISO-HAS cells were treated with siRNA for the negative control (NC) or PDK1 for 6 h. Cell extracts were analyzed by immunoblotting for the indicated proteins. (D) Clonogenic suppression by the knock down of PDK1. ISOS1 cells were transfected with siRNA for NC or PDK1. After 6 h, cells were treated with 1 mM of everolimus and incubated for 12 h. Colony numbers were counted after 10 days. Data represent the means of triplicates with SD indicated. Significance was assessed by Student’s t test. * P < 0.01, EV; everolimus.

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of cyclin D in ISOS-1 and ISO-HAS cells. Although the growth of each cell was not suppressed by MK2206 (Fig. 3A), MK2206 decreased phospho-AKT (Thr308) and phospho-AKT (Ser473) levels, but not cyclin D levels (Fig. 3B). These results indicated that AKT was not necessary for the expression of cyclin D in ISOS-1 and ISO-HAS cells. 3.3. PDK1-dependent cell growth and cyclin D regulation As a downstream signal of PI3K, PDK1 plays a central role in many signal transduction pathways, and activates 20 substrates including AKT, PKC, and S6 kinase. To investigate the possible mechanisms underlying the PDK1-dependent and AKT-independent regulation of cyclin D, we used PDK1 inhibitor OSU-03012 in both cells. OSU-03012 inhibited the growth of each cell line in a dose-dependent manner (Fig. 4A), and decreased the phosphorylation of AKT (Thr308), and the expression of cyclin D in ISOS-1 and ISO-HAS cells (Fig. 4B). However, OSU-03012 more evidently decreased phospho-S6 kinase in ISOS-1 cells rather than in ISOHAS cells, probably reflecting the ability inducing the G1-phase arrest (Fig. 4B). OSU-03012 is a derivative of celecoxib, a cyclooxygenase-2 inhibitor, and is known to be a selective PDK1 kinase inhibitor. However, OSU-03012 was reported to have other effects such as induction of autophagy [17–19]. Therefore, we used siRNA for PDK1 to investigate the dependency of PDK1 on the regulation of cyclin D and cell growth. Although phospho-AKT (Thr308) was slightly reduced in both cells treated with siPDK1, the expressions of cyclin D, phospho-S6, and phospho-4E-BP1 were evidently decreased (Fig. 4C). We used a colony formation assay with siPDK1 to confirm PDK1-dependent growth in both cells. The knock down of PDK1 with siRNA significantly decreased colony numbers below those in the negative control groups treated with or without 1 mM of everolimus in ISOS-1 cells (Fig. 4D). On the other hand, ISO-HAS cells did not form colonies, so we could not evaluate the dependency of PDK1 on clonogenic proliferation. These results suggest that PDK1 may be an attractive therapeutic target for the treatment of angiosarcoma. 4. Discussion Aberrant PI3K signaling commonly occurs in cancer. PI3Ks phosphorylate the D3 position on membrane phosphatidylinositides and proteins containing pleckstrin homology or other lipidbinding domains, and these are recruited and activated on inner cellular membranes. The serine/threonine kinase AKT/PKB, which is activated by PDK1 and the mTORC2 complex, is considered to transduce the major downstream of PI3K signal in cancer. Although several PIK3CA mutations have been reported in angiosarcoma tumors, a relationship has not been identified between the PIK3CA mutational status and pS6K and/or p4EBP-1 by immunohistochemical analysis [7]. The results of the present study showed that the PI3K inhibitor LY294002 and PI3K/mTOR inhibitor NVP-BEZ235 suppressed the growth of angiosarcoma cells (Fig. 1), whereas the RAF inhibitor PLX4720 and MEK inhibitor PD0325901 showed weaker growth inhibition (Supplementary Fig. S1), which is consistent with previous findings in which the PI3K pathway was more important than the MAPK pathway in angiosarcoma [8,9]. AKT regulates cell growth and survival pathways by phosphorylating substrates such as GSK3, Forkhead transcription factors, and TSC2 tumor suppressor protein. The overexpression of AKT1 in an immortalized murine endothelial cell line resulted in benign vascular malformations rather than angiosarcoma [20]. The AKT inhibitor MK2206 did not suppress the growth of ISOS-1 and ISO-HAS cells (Fig. 3A) and also did not regulate cyclin D levels in ISOS-1 and ISO-HAS cells (Fig. 3B).mTOR plays a key role in cell growth and homeostasis [21,22], and the

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activation of its pathway has been reported in angiosarcoma [7,10]. Everolimus, an oral mTOR inhibitor, is a rapamycin derivative agent, which is widely used against renal cell carcinoma. In this study, everolimus inhibited the growth of ISOS-1 and ISOHAS cells, and caused G1 cell-cycle arrest in ISOS-1 and ISO-HAS cells. However, the accumulation of G1-phase cells and deregulation of cyclin D levels by everolimus were less than those by the PI3K inhibitor LY294002 or PI3K/mTOR inhibitor NVP-BEZ235 in ISOS-1 cells (Fig. 2A and E). Therefore, the mTOR inhibitor was not sufficient to inhibit the growth of angiosarcoma cells. Phospho-PDK1 levels have not correlated with phospho-AKT and PIK3CA mutations in many human PIK3CA mutant cancer cell lines [23]. PI3K promotes cancer growth through both AKTdependent and AKT-independent mechanisms [23]. In our results, ISOS-1 and ISO-HAS cells were suppressed by the PI3K inhibitor LY294002 and PI3K/mTOR inhibitor NVP BEZ-235 (Fig. 1), but were not suppressed by the AKT inhibitor MK2206 (Fig. 3A). PDK1 knock down with siRNA suppressed colony numbers and the expression of cyclin D, which indicated a functional dependence on PDK1 in ISOS-1 cells (Fig. 4C and D). Our results suggest that PDK1 inhibitor is promising as an agent for angiosarcoma therapy.

Funding source None declared.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jdermsci.2015. 01.015.

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PDK1 is a potential therapeutic target against angiosarcoma cells.

Angiosarcoma is a rare and aggressive malignant neoplasm of endothelial cells. Recent studies have shown that the mTOR pathway is also aberrantly acti...
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