Targeting Iκb Kinase Β/NF-Κb Signaling in Human Prostate Cancer by a Novel Iκb Kinase Β Inhibitor Cmpda

Published OnlineFirst April 8, 2016; DOI: 10.1158/1535-7163.MCT-15-0999

Small Molecule Therapeutics Molecular Cancer Therapeutics Targeting IkB Kinase b/NF-kB Signaling in Human Prostate Cancer by a Novel IkB Kinase b Inhibitor CmpdA Yanting Zhang1, Rena G. Lapidus1, Peiyan Liu1, Eun Yong Choi1, Samusi Adediran1, Arif Hussain1,2, Xinghuan Wang3, Xuefeng Liu4, and Han C. Dan1,5

Abstract

NF-kB plays an important role in many types of cancer, includ- allel among the respective TMA samples analyzed. IKKb, but not ing prostate cancer, but the role of the upstream kinase of NF-kB, NF-kB, is found to regulate Nanog, which, in turn, modulates the IKKb, in prostate cancer has neither been fully documented nor levels of Oct4, Sox2, Snail, and Slug, indicating an essential role of are there any effective IKKb inhibitors used in clinical settings. IKKb in regulating CSCs and EMT. The novel IKKb inhibitor Here, we have shown that IKKb activity is mediated by multiple CmpdA inhibits constitutively activated IKKb/NF-kB signaling, kinases including IKKa in human prostate cancer cell lines that leading to induction of apoptosis and inhibition of proliferation, express activated IKKb. IHC analysis (IHC) of human prostate migration, and stemness in these cells. CmpdA also signiﬁcantly cancer tissue microarrays (TMA) demonstrates that phosphory- inhibits tumor growth in xenografts without causing apparent lation of IKKa/b within its activation loop gradually increases in vivo toxicity. Furthermore, CmpdA and docetaxel act synergis- in low to higher stage tumors as compared with normal tissue. tically to inhibit proliferation of prostate cancer cells. These results The expression of cell proliferation and survival markers (Ki-67, indicate that IKKb plays a pivotal role in prostate cancer, and Survivin) and epithelial-to-mesenchymal transition (EMT) mar- targeting IKKb, including in combination with docetaxel, may be kers (Slug, Snail), as well as cancer stem cell (CSC)-related a potentially useful strategy for treating advanced prostate cancer. transcription factors (Nanog, Sox2, Oct-4), also increase in par- Mol Cancer Ther; 1–11. 2016 AACR.

Introduction improve treatment outcomes, identification of relevant signaling pathways associated with the development of CRPC and resis- Prostate cancer ranks as the second most common cause of tance to cytotoxic agents remains a major challenge. cancer-related deaths among men in the western world (1). The The PI3K signaling pathway plays a critical role in many standard treatment for metastatic prostate cancer is androgen- physiologic cellular processes as well as in cancer. Activated PI3K deprivation therapy (ADT), but while initially effective in induc- phosphorylates PtdIns(4,5)P2 to produce phosphoinositide ing a clinical response, invariably fails within 2 years in most PtdIns(3,4,5)P3 that, in turn, recruits PDK1 and Akt to the plasma patients, resulting in castration-resistant prostate cancer (CRPC). membrane where Akt is phosphorylated and activated by PDK1. Several treatment options are currently available for CRPC The tumor suppressor gene PTEN is a phosphatase that depho- patients, including second-line hormonal therapy, a dendritic sphorylates PtdIns(3,4,5)P3, thus decreasing Akt activity (6). cell-based vaccine (sipuleucel-T), bone-targeted agents (radi- Inactivation of PTEN mutations is common in cancer, resulting um-223), and cytotoxins such as docetaxel and cabazitaxel. How- in constitutive activation of Akt. One of the major downstream ever, these treatments ultimately provide limited benefit due to substrates of Akt is TSC2, which is phosphorylated to activate the inability to provide a long-lasting response (2–5). Thus, to the mTOR, another important tumorigenesis-promoting kinase (7–10). PTEN mutations have been identified in over 50% of tumor samples from patients with CRPC. The cumulative evi- 1Marlene and Stewart Greenebaum Cancer Center, University of Mary- dence indicates that loss of PTEN function and subsequent 2 land School of Medicine, Baltimore, Maryland. Baltimore VA Medical activation of Akt and mTOR are critical events in the progression Center, Baltimore, Maryland. 3Department of Urology, Zhongnan Hos- pital of Wuhan University, Wuhan, Hubei, P.R. China. 4Department of of human prostate cancer (11, 12). Pathology, Georgetown University Medical Center, Washington, DC. The NF-kB family is composed of five members: RelA (p65), 5 Department of Pathology, University of Maryland School of Medicine, RelB, c-Rel, p50/p105 (NF-kB1), and p52/p100 (NF-kB2). NF-kB Baltimore, Maryland. signaling is activated by a variety of inflammatory mediators and Note: Supplementary data for this article are available at Molecular Cancer growth factors through canonical and noncanonical pathways Therapeutics Online (http://mct.aacrjournals.org/). (13, 14). In most signaling cascades, NF-kB is activated by the Corresponding Author: Han C. Dan, Marlene and Stewart Greenebaum Cancer canonical pathway that utilizes the IKK complex that contains Center, University of Maryland School of Medicine, Baltimore, MD 21201. Phone: IKKa, IKKb, and IKKg. IKKa and IKKb drive the catalytic activity of 410-328-0372; Fax: 341-576-873; E-mail: [email protected] IKK, with IKKb playing a dominant role in inflammatory-medi- doi: 10.1158/1535-7163.MCT-15-0999 ated pathways. Normally, p50 and p65 NF-kB heterodimers are 2016 American Association for Cancer Research. sequestered in the cytoplasm in an inactive state by IkBa. Upon

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stimulation, the IkB kinase (IKK) complex is activated, leading to minutes, samples containing 20 to 50 mgprotein/lanewere IkBa phosphorylation, which results in ubiquitination and pro- resolved by SDS-PAGE, transferred to Pure Nitrocellulose Mem- teasomal degradation of IkBa and subsequent p50 and p65 brane (Bio-Rad), blocked in 5% nonfat milk, and then blotted heterodimer translocation to the nucleus. Dysregulation of NF- with the indicated antibodies. kB activity is associated with numerous cancers (13, 15), and can induce gene expression to regulate different cellular processes, RNAi including cell proliferation, survival, invasion, metastasis, and siRNA SMARTpool Raptor, IKKa, IKKb, NF-kB p65, and Nanog resistance to chemotherapy (13–16). were from Dharmacon. Each represents four pooled SMART- A number of studies have shown that NF-kB signaling plays a selected siRNA duplexes that target the indicated genes. PC3 cells pivotal role in prostate cancer (17–19). Clinically, prostate cancer were transfected with SMARTpool IKKb, p65, or nonspecific patients with elevated NF-kB have a worse prognosis. The extent control siRNAs using DharmaFECT 1 reagent (Dharmacon) of nuclear NF-kB p65 staining has been shown to correlate with according to the manufacturer's instructions. In brief, a final tumor grade in prostate cancer (20). In addition, it has been concentration of 20 nmol of siRNA was used to transfect cells reported that NF-kB signaling is upregulated in CRPC patients and for 48 to 72 hours. correlates with disease progression (21–24). We have previously shown that both mTORC1 and NF-kB activity are upregulated Cell proliferation assay downstream of Akt in PTEN null prostate cancer cells, and Cell proliferation was measured by MTS assay using the Cell- mTORC1 interacts with the IKK complex to promote induction Titer 96 Aqueous ONE Solution kit (Promega). Briefly, cells were of NF-kB (25, 26). In the present study, we hypothesize that IKKb, seeded into 96-well plates at a density of 5 104 cells/mL for 24 the upstream regulator of NF-kB, is an essential driver in prostate hours, then the culture media were replaced with fresh media cancer. We carried out both in vitro and in vivo studies to determine containing the indicated concentrations of CmpdA or vehicle the role of IKKb/NF-kB in regulating prostate cancer tumorigen- control (DMSO). After incubation for an additional 48 hours, esis. In addition, we evaluated the targeting of IKKb by the novel MTS reagent (20 mL) was added to each well and incubated at IKKb inhibitor CmpdA in prostate cancer cells expressing activat- 37 C for 1 to 4 hours. Absorbance at 490 nm was measured using ed IKKb. Our results suggest that IKKb may be an important target a microplate reader (Bio-Rad). Three independent experiments in prostate cancer, particularly in the context of functional PTEN were performed, each in triplicate. deficiency. Caspase-3/7 activity assay Materials and Methods Cells were plated in triplicate at 4 103 cells/per well in white- walled 96-well plates (Becton Dickinson). Cells were transfected Chemical reagents with siRNA as described above and treated with the IKK inhibitor The Akt inhibitor Perifosine (KRX-0401) was purchased from and/or docetaxel as indicated. Caspase-3/7 activity was measured Selleck Chemical. Docetaxel was purchased from Cell Signaling at 48 hours post-transfection using the Caspase-Glo 3/7 assay Technology. The IKKb inhibitor, CmpdA, was kindly provided by (Promega) according to the manufacturer's instructions. The Dr. Albert Baldwin, University of North Carolina (Chapel Hill, Caspase-Glo 3/7 assay uses a caspase-3/7 tetrapeptide DEVD NC). Antibodies against IKKa, IKKb, and Akt were obtained from substrate that produces a luminescent signal on cleavage. Relative Upstate Biotechnology. Antibodies for IHC, including Ki-67, light units were measured on an Lmax Microplate Luminometer cleaved caspase-3, p-IKKa/b-S177/S181, Slug, and Snail were (Molecular Devices). from Abcam. Horseradish peroxidase-labeled anti-mouse and anti-rabbit secondary antibodies were from Santa Cruz Biotech- Colony formation assays nology. Antibodies for Oct4 (CST-2750), Sox2 (CST-3728), p65 One thousand cells were seeded into 6-well plates and incu- (CST-8242), p-p65 (CST-3033), Survivin (CST-2808), p-Akt- bated for 5 or 7 days after the appropriate treatment. The colonies S473 (CST-4058), GAPDH (CST-5174), as well as any additional were stained with crystal violet (0.5% w/v) and images taken antibodies were from Cell Signaling Technology. using a scanner (Epson).

Cell lines and cell culture Analysis of cell cycle and apoptosis via flow cytometry Prostate cancer cell lines, PC3 and Du145, were purchased from Cell-cycle analysis was performed as described previously (27). ATCC in 2013, and no authentication was performed in the In brief, cells were fixed with 70% cold ethanol at 20 C for at least laboratory. All cells were maintained in DMEM supplemented 1.5 hours and stained with PBS containing 50 mg/mL propidium with 10% FBS, 2 mmol glutamine, and 100 U/mL penicillin and iodide (PI) and 30 mg/ml RNase A, and the samples analyzed for streptomycin. All the cell lines were meticulously passaged and DNA content and apoptotic cells (sub-G0/G1 fraction) via a flow tested for mycoplasma contamination. cytometer (FACS Canto; Becton Dickinson).

Cell lysis and Western blotting Cell migration assay Cells were grown in 100-mm dishes, rinsed twice with cold Scratch wound-healing assay was performed to determine cell PBS, and then lysed on ice for 20 minutes in 1 mL of lysis buffer migration using confluent cultures (80%–90% confluence). Cells [40 mmol/L HEPES (pH 7.5), 120 mmol/L NaCl, 1 mmol were photographed under a microscope at 0, 24, and 48 hours. EDTA, 10 mmol pyrophosphate, 10 mmol glycerophosphate, 50 mmol NaF, 0.5 mmol orthovanadate, and EDTA-free pro- Real-time cell migration assay tease inhibitors (Roche Applied Science)] containing 1% Triton Seventy-five thousand cells were plated in serum-free media in X-100 or 0.3% CHAPS. After centrifugation at 13,000 g for 15 the upper chamber of a CIM plate after serum starvation for 18

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hours and CmpdA (2 or 5 mmol/L) or vehicle control were added depletion of mTORC1 (by mTOR or Raptor knockdown) simultaneously. The lower chamber was filled with either serum- decreased the activity of both IKKa and IKKb (26), suggesting free media (negative control) or full media (DMED with 10% that mTORC1 regulates IKKa and IKKb downstream of Akt FCS). Cells (with or without treatment) were allowed to equili- (Supplementary Fig. S1A). Decreased phosphorylation of S6K, brate at room temperature for 1 hour and then incubated at 37C p65, and IkBa occurred after knockdown of IKKa (Supplemen- for 48 hours in an Xcelligence DP module. taryFig.S1D).Importantly,phosphorylationofIKKb was also dramatically reduced in a dose-dependent manner upon IKKa Tumor xenograft study knockdown (Supplementary Fig. S1C). Taken together, these The Translational Laboratory Shared Services (TLSS) at the results suggest that IKKa can affect IKKb through mTORC1- University of Maryland Greenebaum Cancer Center (Baltimore, dependent and independent mechanisms (Supplementary Fig. MD) performed the in vivo studies using 8-week-old male nu/nu S1A and S1C). In addition, knockdown of IKKb impairs the mice (Harlan Sprague Dawley). A total of 5 106 PC3 cells were phosphorylation of both IKKa and p65, yet has no significant mixed with 50 mL Matrigel and injected into the flanks of nude effects on S6K phosphorylation. Thus, IKKb appears to activate mice. Nine days after cell injection, mice were sorted into three NF-kB downstream of IKKa and mTORC1, and is, itself, acti- balanced groups. CmpdA (10 or 15 mg/kg) or vehicle control (10% vated by Akt, mTORC1, and IKKa in PC3 cells (Supplementary dimethyl sulfoxide in saline) was given intraperitoneally (IP) twice Fig. S1A and S1D). a week for 3 weeks. The growth of the tumors was measured with b k electronic calipers twice per week for 3 weeks. Tumor volumes were Depletion of IKK /NF- B signaling inhibits cell proliferation calculated using the equation Vol ¼ (L W2)/2 where L is the and migration and leads to apoptosis in PC3 cells longer dimension and W is the shorter dimension. The tumors were To determine whether the IKKb/NF-kB pathway plays a role then harvested for subsequent analysis. in tumorigenesis, we evaluated the effects of IKKb and NF-kB knockdown on cell proliferation, survival, and migration. The Tissue microarray results showed that knockdown of either IKKb or p65 leads to Prostate cancer TMA arrays T191 and PR243B were obtained inhibition of cell proliferation, with a greater reduction in cell from US BioMax, Inc. proliferation observed after IKKb knockdown, as compared with p65 (Fig. 1A). Reduction in IKKb or p65 expression caused adramaticincreaseinapoptosisaswellasasignificant decrease IHC analysis in PC3 cell colony formation, with greater effects on these All IHC analyses were conducted by the Pathology Core Facility parameters noted with IKKb knockdown (Fig 1B and 1C). PC3 at the Baltimore VA Medical Center. cell migration was also strongly suppressed after either IKKb or p65 knockdown (Fig. 1D) in line with the decrease of cell Statistical analysis proliferation (Fig. 1E). Consistent with the role of IKKb/p65 Data are presented as mean SD. Statistical analysis was signaling in cell survival, Western blot analysis revealed that performed using GraphPad Prism version 4.0 (GraphPad Soft- knockdown of either IKKb or p65 resulted in decreased expres- ware Inc.). One-way ANOVA followed by Dunnett's multiple sion of Survivin and enhanced cleavage of caspase-3 (Fig. 1E). fi comparisons test was used to analyze the statistical signi cance Interestingly, epithelial-to-mesenchymal transition (EMT) mar- of 3 or more groups. P values <0.05 were considered to be kers were differentially affected by IKKb and p65. IKKb knock- fi statistically signi cant. down dramatically reduced the expression of both Slug and Snail, whereas knockdown of p65 decreased only Snail expres- Results sion (Fig. 1E). Taken together, the above studies demonstrate IKKb activity is regulated by Akt, mTORC1, and IKKa in that IKKb and p65 are important in regulating key biologic prostate cancer PC3 cells properties of PC3 cells, with IKKb, in particular, playing a more Our previous studies showed that both mTORC1 and NF-kB prominent role in these processes. activity were upregulated in AR-negative PTEN null PC3 prostate cancer cells (25, 26). In these cells, the IKK complex Knockdown of IKKb/NF-kB inhibits stemness in PC3 cells associates with the mTORC1 complex downstream of Akt. To determine whether the IKKb/NF-kB pathway plays a role in Moreover, IKKa enhances mTORC1 activity, while mTORC1 modulating stemness in prostate cancer cells, the expression promotes IKKa and IKKb activity to upregulate NF-kBfunction patterns of the transcription factors Nanog, Oct-4, and Sox2 were in a positive feedback loop (25, 26). These findings suggested examined after knockdown of IKKb or p65 NF-kB. Knockdown of that IKKb could be a downstream target of Akt, mTORC1, and IKKb decreased Nanog, Oct-4, and Sox2 levels, whereas knock- IKKa in PC3 cells (Supplementary Fig. S1A). To test this model, down of NF-kB caused only minimal reduction of Sox2 and had we first employed a pan Akt inhibitor, perifosine, that simul- no effects on Oct4 and Nanog expression (Fig. 2A). Previous taneously blocks the activity of all three isoforms of Akt (28, reports showed that Nanog is upstream of Oct-4 and Sox2 29). PC3 cells were treated with different concentrations of (30, 31), as well as Snail and Slug. Knockdown of Nanog perifosine for 2 hours and phosphorylation of Akt, IKK (both decreased the levels of Oct-4, Sox2, Snail, and Slug in PC3 cells IKKa and IKKb), S6K (mTOR target), and IkBa and p65 (IKK but had no effect on IKKb expression (Fig. 2B). Similar results were substrates) was determined by Western blot analysis. As shown observed by immunofluorescence (Fig. 2C). Thus, IKKb, in par- in Fig. S1B, perifosine dramatically reduced Akt phosphoryla- ticular, appears to be relevant in the regulation of several mod- tion,aswellasthatofS6K,IKKa/b,IkBa, and p65, confirming ulators of EMT, primarily in a NF-kB–independent manner that both mTORC1 and IKK/NF-kB signaling pathways are (Fig. 2D). Furthermore, knockdown of IKKb or NF-kB can sig- downstream targets of Akt. We previously demonstrated that nificantly impair the ability of PC3 cells to form tumor spheres

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Figure 1. Knockdown of IKKb impairs cell proliferation, survival, and migration. A, PC3 cells were transfected with control, IKKb, or p65 siRNA for 72 hours and cell proliferation was determined by MTT assay. The experiments were repeated three times in triplicate (, P < 0.05; , P < 0.01; , P < 0.001). B, cells were treated as described in Fig. 1A for 72 hours and caspase activity was measured. The experiments were repeated three times in triplicate (, P < 0.01; , P < 0.001). C, cells were transfected with control, IKKb, or p65 siRNA for 24 hours followed by seeding of 1,000 cells in 6-well plates, then incubation for 5 days, followed by colony staining. D, cells were transfected with control, IKKb, or p65 siRNA for 48 hours. Scratches were made on cell monolayers and wound closure was monitored at 0, 24, and 48 hours. The experiments were repeated three times. E, cells were transfected with control, IKKb, or p65 siRNA for 48 hours before planted in 96-well plates and cell proliferation was determined by MTT assay at 0, 24, and 48 hours (, P < 0.05; , P < 0.01). F, cells were transfected with control, IKKb, or p65 siRNA for 72 hours and the expressions of IKKb, p65, survivin, cleaved caspase-3, Slug, and Snail were determined by Western blot analysis. GAPDH was used as a loading control. The experiments were repeated three times.

(Fig. 2E). Moreover, when PC3 cells are sorted into CD44hi and Effects of the novel IKKb inhibitor CmpdA on PC3 CD44lo subpopulations, the enhanced phosphorylation of IKK and DU145 cells and NF-kB in the former subpopulation further suggests that CmpdA is a novel selective low-molecular-weight inhibitor IKKb/NF-kB signaling is associated with a cancer stem-like cell of IKKb (32). Treatment of PC3 cells with 2 mmol/L CmpdA (CSLC) phenotype (Fig. 2F). for 0.5 to 2 hours results in prominent inhibition of phosphorylation of IKKb without any effects on IKKa phosphorylation. IKK phosphorylation is consistent with expression of EMT- and Consistently, IkBa and p65 phosphorylation deceased upon CSC-related proteins in prostate cancer patients CmpdA treatment (Fig. 3A). CmpdA caused rapid and complete Next, tissue microarrays derived from patient tumor samples inhibition of IkBa phosphorylation, whereas the dynamics of p65 were examined via IHC. Phosphorylation of IKKa/b and the inhibition were more protracted with maximal inhibition of the expressions of Ki-67 and Survivin gradually increased from latter occurring at 24 hours (Fig. 3B). Interestingly, some resur- normal tissue to T2N0M0 to T3N0M1 to T4N1M1 tumors, gence in IkBa and p65 phosphorylation 48 hours after treatment suggesting that increased IKK activity is associated with higher may in part be due to the short half-life of CmpdA and/or clinical stages of prostate cancer (Supplementary Fig. S2, left). reﬂect an escape mechanism (Fig. 3B). Furthermore, the expres- Furthermore, the expressions of Snail and Slug, as well as sion of Survivin is suppressed upon 48 hours CmpdA treatment Nanog, Oct-4, and Sox2 also increased in parallel from normal (Fig. 3B). CmpdA also induced caspase-3 cleavage in a dose- and tissue to low and higher stage tumors (Supplementary Fig. S2, time-dependent manner (Fig. 3B). In addition, CmpdA sup- right), suggesting that IKKb and related downstream signaling presses NF-kB promoter-reporter activity in a dose-dependent may be particularly relevant in the more advanced stages of manner (Fig. 3C). In terms of potential anticancer effects, CmpdA prostate cancer. induces cellular caspase-3/7 activity (Fig. 3D) and inhibits cell

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Figure 2. Knockdown of IKKb inhibits stemness via Nanog, Oct-4, and Sox2 in PC3 cells. A, cells were transfected with control, IKKb, or p65 siRNA for 72 hours and the expressions of IKKb, p65, Nanog, Oct4, and Sox2 were determined by Western blot analysis. GAPDH was used for a loading control. B, cells were transfected with control or Nanog siRNA for 72 hours and the expressions of IKKb, Nanog, Oct4, Sox2, Snail, and Slug were determined by Western blot analysis. GAPDH was used for a loading control. C, cells were transfected with control, IKKb, or Nanog siRNA for 72 hours and the expressions of IKKb and Nanog determined by immunoﬂuorescence (IF). D, proposed model of IKKb regulation of CSC and EMT through NF-kB–dependent and -independent mechanisms. E, cells were transfected with control, IKKb, p65, or Nanog siRNA, and tumor sphere formation was monitored and counted (, P < 0.01; , P < 0.001). F, cells were sorted via ﬂow cytometry using CD44 antibody, cultured and then analyzed by Western blot analysis with the indicated antibodies.

proliferation (Fig. 3E). A dose-dependent increase in the sub-G0/ Phosphorylation of both NF-kB and IkBa was inhibited by G1 peak on ﬂow cytometry corroborates the proapoptotic effects CmpdA in a dose-and time- dependent manner (Supplementary of CmpdA on PC3 cells (Fig. 3F). In addition, at higher concen- Fig. S3A and S3B). Similar to PC3 cells, CmpdA dramatically trations, CmpdA signiﬁcantly prevents PC3 cells from entering the inhibited DU145 cell proliferation, increased cellular caspase-3/7 S phase, and also gradually decreases the percentage of cells with activity, decreased colony formation, induced cell apoptosis, DNA content > 4N (Fig. 3F), suggesting that it inhibits G1/S suppressed expression of Survivin, and increased expression of transition and mitosis in PC3 cells. Consistent with its antipro- cleaved caspase-3 (Supplementary Fig. S3C–S3H). liferative and proapoptotic properties, CpmdA inhibits colony formation of PC3 cells (Fig. 3G). CmpdA suppresses tumorigenesis in vivo We extended the above studies to another AR-negative prostate ArecentstudydemonstratedthatCmpdA(10mg/kg)inhibited cancer cell line, DU145, that also possesses high basal levels of NF- K-Ras induced lung tumors in a mouse model (34). We evaluated kB activity but has wild-type PTEN (33). DU145 cells have high the tolerability and antitumor activity of CmpdA in vivo using levels of phosphorylated p65 and IkBa, suggesting that IKK is mouse xenografts. When tumors reached approximately constitutively active in these cells (Supplementary Fig. S3A). We 200 mm3 after PC3 cell inoculations, mice were randomized to evaluated the antitumor activity of CmpdA in DU145 cells. one of three treatment groups: vehicle control (n ¼ 7), 10 kg/mg

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Figure 3. CmpdA inhibits IKKb/NF-kB signaling, cell proliferation, and survival in PC3 cells. A, PC3 cells were treated with vehicle control or CmpdA (2 mmol/L) for 0.5 to 2 hours and phosphorylation status of IKKa/b,IkBa, and p65 was analyzed by Western blot analysis. B, PC3 cells were treated with vehicle control or CmpdA (2 to 10 mmol/L) for 0.5 to 48 hours and phosphorylation status of IkBa and p65 and levels of survivin and cleaved caspase-3 were analyzed by Western blot analysis. C, PC3 cells were transfected with NF-kB reporter and Renilla reporter control for 24 hours and treated with CmpdA for an additional 24 hours. Cells were harvested and an NF-kB reporter assay was performed. The experiments were repeated three times. D, cells were treated with CmpdA for 48 hours and cell apoptosis was determined by measuring caspase activity. The results are representative of three independent experiments (, P < 0.05; , P < 0.01; , P < 0.001). E, cells were treated with CmpdA for 48 hours and cell proliferation was determined by MTT assay. The results are representative of three independent experiments (, P < 0.05; , P < 0.001). F, apoptosis was determined via flow cytometry. PC3 cells were cultured with increasing concentrations of CmpdA (2, 5, and 10 mmol/L) for 72 hours. Cells were collected and washed twice with ice-cold PBS, fixed with 70% cold ethanol at 20C for 1.5 hours, and stained with 1 PBS containing 50 mg/mL propidium iodide (PI) and 30 mg/mL RNase A, and the samples were analyzed for DNA content and apoptotic cells via flow cytometry (, P < 0.05; , P < 0.01; , P < 0.001). G, cells were seeded in 6-well plates, incubated with CmpdA for 7 days, and then stained for colony formation. The results are representative of three independent experiments.

(n ¼ 6) or 15 kg/mg CmpdA (n ¼ 6). The mice were treated via data are consistent with the notion that inhibition of IKKb can intraperitonealinjection(IP)twiceaweekfor3weeks.Bothdoses lead to antitumor effects in an in vivo model of prostate cancer. of CmpdA were tolerable as shown by the lack of weight loss among the treated mice for the duration of the study (Fig. 4A). Inhibition of IKKb/Nanog signaling as well as EMT, migration, As a single agent, CmpdA has modest but consistent inhibitory and stemness by CmpdA effects on PC3 tumor growth in vivo, as determined by relative CmpdA impaired cell migration in a dose-dependent manner tumorvolumeandtumorweightmeasurements(Fig.4Band4C). in a wound-healing assay (Fig. 5A). Similarly, it inhibited PC3 cell Immunoﬂuorescence assays demonstrated that CmpdA inhib- migration in the xCELLigence real time cell system as noted by a ited phosphorylation of IKK in the xenograft tumors (Fig. 4D). change in the cell index over time (Fig. 5B). Similar effects of Furthermore, IHC staining revealed that Ki-67 and Survivin CmpdA on the migratory characteristics were observed in DU145 expression were reduced dramatically while cleaved caspase-3 cells (Supplementary Fig. S4A–S4C). CmpdA also inhibits PC3 was increased in tumors from CmpdA-treated mice compared to cell tumor sphere formation (Fig. 5C), and the expression of EMT tumors from vehicle-treated mice (Fig. 4E). Taken together, these markers including Snail and Slug, as well as transcription factors

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Figure 4. CmpdA suppresses xenograft tumor formation and induces apoptosis in tumors. A, mice inoculated with PC3 cells were treated with different doses of CmpdA. Mouse body weights were monitored twice per week for three weeks (left) and final body weights were determined at the time of sacrifice (right). B, tumor growth curves based on tumor volumes measured twice weekly (, P < 0.05; , P < 0.01; , P < 0.001). C, tumors were photographed and weights recorded at the time of sacrifice (, P < 0.05; , P < 0.01). D, xenograft tumors harvested from vehicle- or CmpdA-treated mice were stained with p-IKKa/b. E, xenograft tumors harvested from vehicle- or CmpdA-treated mice were stained with Ki-67, survivin, or cleaved caspase-3 antibodies, and representative images are shown.

Nanog, Oct-4, and Sox2 (Fig. 5D). The expressions of other cancer bination of CmpdA and Docetaxel. Using the combination index stem cell (CSC)-related genes were also detected. CmpdA (CI) method of Chou and Talalay (35, 36), we tested the effects of decreased expression of Gli-1 while having no effect on b-catenin CmpdA and docetaxel combinations on PC3 and DU145 cells. or Notch. Meanwhile, the expression of Snail, Slug, Nanog, Sox2, The CI values were determined using the CalcuSyn v2.0 software and CD44 were observed in control groups of xenograft tumors that provides quantitative definition for additive (CI ¼ 1), syn- especially on the margin of the tumors and were significantly ergistic (CI < 1), and antagonistic (CI > 1) effects of drug combi- decreased in CmpdA treated tumors (Fig. 5E). nations (35, 36). The CI values were less than 1 with the CmpdA/ docetaxel combination with respect to both PC3 and DU145 cells CmpdA synergizes with docetaxel to induce apoptosis in PC3 (Fig. 6A and E), indicating synergistic antiproliferative effects and DU145 cells when the two agents were combined. CmpdA in combination The above series of studies demonstrate that CmpdA as single with docetaxel also induced caspase activation and caspase-3 agent has significant antitumor activity in prostate cancer cells that cleavage in a dose-dependent manner in both PC3 (Fig. 6B–D) have constitutive IKKb activation. Docetaxel is an FDA-approved and DU145 cells (Fig. 6F–H). Thus, the combination of CmpdA chemotherapy drug used to treat recurrent or metastatic castra- and docetaxel has strong synergistic antiproliferative activity and tion-resistant prostate cancer; however, its efficacy is very limited induces apoptosis in prostate cancer cells. in patients due to acquired resistance (4, 5). It is important to improve the efficacy of docetaxel by combining it with other single- or multiple-pathway inhibitors. We wanted to determine Discussion whether enhanced antitumor inhibition could occur by dual Activation of Akt can lead to recruitment of downstream path- targeting of IKKb and the microtubular network through a com- ways that are important in modulating proliferative, anabolic,

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Zhang et al.

CmpdA CmpdA A CmpdA CmpdA CmpdA D Control 5 μmol/L 10 μmol/L μ Control 2 mol/L 5 μmol/L 10 μmol/L 600 CmpdA 12 day 0 h 6 day 400

200 48 h (>50 μ mol/L)

Sphere number 0 μ

12 day 0 5 10 mol/L

B E Control CmpdA treatment

DMSO

CmpdA 2 μmol/L Slug Slug CmpdA 5 μmol/L SFM Cell index

Time (in hours) Snail Snail

C 24 h 48 h CmpdA 0 2 5 10 2 5 10 μmol/L β-Catenin

Nortch CD44 CD44

Gli-1

Nanog

Oct4 Nanog Nanog

Sox2

Slug

Snail Sox2 Sox2 GAPDH

Figure 5. Suppression of cell migration and stemness by CmpdA. A, cells were treated with vehicle control or CmpdA, and scratches were made on cell monolayers. Wound closure was monitored at 0 and 48 hours. The experiments were repeated three times. B, cells were treated with vehicle control or CmpdA and migration was monitored using the xCELLigence System real-time cell analyzer instrument. C, cells were treated with CmpdA and tumor sphere formation was monitored; representative images are shown (, P < 0.05; , P < 0.01). D, cells were treated with different doses of CmpdA (0–48 hours), lysed, and analyzed by Western blot analysis. The results are representative of three independent experiments. E, xenograft tumors harvested from vehicle- or CmpdA-treated mice were stained with Slug, Snail, Oct-4, Nanog, and Sox2 antibodies, and representative images are shown.

and antiapoptotic programs in cancer cells (6). Considerable the Akt/mTORC1 signaling cascade (25, 26). In the present efforts are being made to target Akt for the treatment of cancer. study, we studied the more detailed regulatory mechanisms However, to date, strategies to target Akt as an anticancer therapy whereby IKK/NF-kB is activated downstream of Akt in prostate have met limited success due to toxicity and recruitment of cancer cells. We now demonstrate that IKKb, the direct kinase of compensatory mechanisms (29, 37, 38). One of the critical NF-kB, is activated not only by Akt and mTORC1, but also by downstream targets of Akt is mTORC1, which plays an important IKKa. It should be noted that IKKa regulation of IKKb may role in controlling Akt-induced oncogenic events (6). However, involve mTORC1-dependent and/or independent mechanisms; mTOR inhibitors, such as rapamycin, and their derivatives show the latter may be particularly relevant in prostate cancer cells modest beneﬁts in prostate cancer (39, 40). Hence, identiﬁcation such as DU145 that have wild-type PTEN and hence, unlike of other targetable oncogenic pathways that are turned on by loss PC3 cells, do not have constitutive activation of Akt/mTOR of PTEN and activation of Akt and mTOR in prostate cancer is (Fig. 1A). Regardless of the upstream pathways that may lead to important to for their potential therapeutic effects in this disease. IKKb activation, our studies demonstrate that knockdown of NF-kB and its upstream kinase IKKb are upregulated in IKKb inhibits proliferation and migration, and induces apo- prostate cancer, especially in a metastatic setting (17, 21, 23, ptosis in prostate cancer cells. Mechanistically, knockdown of 24). We previously reported that IKK/NF-kB signaling is acti- IKKb or NF-kB decreases the expression of Survivin, an impor- vated by Akt and mTOR in PC3 prostate cancer cells, which tant antiapoptotic protein and NF-kB target gene, and induces suggests that IKK/NF-kBisanimportantdownstreamtargetof caspase-3 cleavage.

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IkB Kinase b in Human Prostate Cancer

A BCControl DX 1 nmol/L DX 5 nmol/L PC3

Combination: 1 2 3 4 5 6 3 1.00 2 0.75 CA 0.4 μmol/L CA 0.4 + DX 1 CA 0.4 + DX 5 1 0.50 Caspase activity 0 0.25 DX 0 0 0 0 1 1 1 1

Cell proliferation CA 0 0.4 2 5 0 0.4 2 5 0.00 μ μ μ μ CA 0 mol/L CA 0.4 mol/L CA 2 mol/L CA 5 mol/L CA 2 μmol/L CA 2 + DX 1 CA 2 + DX 5 DX 0 1 5 0 1 5 0 1 5 0 1 5 nmol/L

Normalized isobologram Mixture-Algebraic estimate 1.0 1.2 D DX 0 0 0 1 1 1 nmol/L CA 0 2 5 0 2 5 μmol/L 0.8 0.9 P-p65 0.6 CA 5 μmol/L CA 5 + DX 1 CA 5 + DX 5 0.6 P65 0.4 CmpdA

CI ± 1.96 s.d. Cleaved 0.3 0.2 caspase-3

0 0 GAPDH 0 0.2 0.4 0.6 0.8 1.0 0 0.2 0.4 0.6 0.8 1.0 Docetaxcel Fractional effect

EFGDU145 Control DX 1 nmol/L DX 5 nmol/L Combination: 1 2 3 4 5 6 1.00 3

0.75 2 CA 0.4 μmol/L CA 0.4 + DX 1 CA 0.4 + DX 5 0.50 1 Caspase activity 0.25 0

Cell proliferation DX 0 0 0 0 1 1 1 1 CA 0 0.4 2 5 0 0.4 2 5 0.00 CA 0 μmol/L CA 0.4 μmol/L CA 2 μmol/L CA 5 μmol/L CA 2 μmol/L CA 2 + DX 1 CA 2 + DX 5 DX 0 1 5 0 1 5 0 1 5 0 1 5 nmol/L

Normalized isobologram Mixture-Algebraic estimate H 1.0 1.0 DX 0 0 0 1 1 1 nmol/L CA 0 2 5 0 2 5 μmol/L 0.8 0.8 P-p65

0.6 0.6 CA 5 μmol/L CA 5 + DX 1 CA 5 + DX 5 P65

0.4 0.4 Cleaved CmpdA

CI ± 1.96 s.d. caspase-3 0.2 0.2 GAPDH 0 0 0 0.2 0.4 0.6 0.8 1.0 0 0.2 0.4 0.6 0.8 1.0 Docetaxcel Fractional effect

Figure 6. Studies with docetaxel and CmpdA combinations. A and E, PC3 (A) and DU145 (E) cells were treated with vehicle control, CmpdA (CA), docetaxel (DX), or a combination of the two, and cell proliferation was assessed by MTT assay. Also shown are the calculated combination index (CI) values (, P < 0.05; , P < 0.01; , P < 0.001). B and F, PC3 (B) and DU145 (F) cells were treated as in A and E for 48 hours and cell morphology was assessed. C and G, PC3 (C) and DU145 (G) cells were treated as in A and E for 48 hours and caspase activity was measured (, P < 0.05; , P < 0.01). D and H, PC3 (D) and DU145 (H) cells were treated as in A and E for 48 hours and phosphorylation status of p65 as well as cleavage of caspase-3 were determined by Western blot analysis. The experiments were repeated three times.

Increasing evidence implicates cancer stem cells (CSC) in likely via NF-kB–independent mechanisms. Thus, IKKb may be cancer initiation and growth, and resistance to anticancer therapy. important in regulating CSC and EMT in some forms of prostate Wnt, b-catenin, and Notch, as well as certain embryonic stem cell cancer. self-renewal genes such as NANOG, Oct-4, and Sox2, among It should also be noted that compared with knockdown of others, have been shown to play important roles in regulation p65, knockdown of IKKb causes increased induction of apoptosis of some of the biologic properties of CSC. Elevated Nanog is (Fig. 1B), decreased protein levels of Slug (Fig. 1E), Nanog, Oct-4, associated with poorer outcomes in several cancers (11, 30, 31, and Nanog (Fig. 2A), and more impairments of tumor sphere 41–42). Depletion of Nanog results in decreased expressions of formation (Fig. 2E). These data indicate that IKKb modulates the Sox2, Oct-4, Snail, and Slug, and impairs the CSC and EMT malignance of prostate cancer most likely through NF-kB–depen- phenotypes (38). For the ﬁrst time, our data show that IKKb dent and -independent mechanisms, thus demonstrating its regulates Nanog, as well as Sox2, Oct-4, Snail, and Slug, most potential as a critical therapeutic target in prostate cancer.

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Several IKKb inhibitors have been explored as potential ther- profiles, particularly in the subgroups of advanced prostate cancer apeutics in recent years. However, to date, no IKKb inhibitor has patients in whom such pathways may be activated. been successfully developed for clinical use, mainly due to toxicity – fi (43 48). Thus, there remains a signi cant need to identify novel Disclosure of Potential Conflicts of Interest IKKb inhibitors that can be safely exploited clinically. The novel No potential conflicts of interest were disclosed. IKKb-specific inhibitor CmpdA is a competitive ATP inhibitor that downregulates IKKb activity and inhibits IkBa phosphorylation. Authors' Contributions CmpdA has been shown to suppress cell proliferation and sen- Conception and design: Y. Zhang, H.C. Dan sitize several cancer cell lines to chemotherapy in vitro (49). A Development of methodology: Y. Zhang, X. Wang, H.C. Dan recent study showed that CmpdA effectively inhibited K-ras Acquisition of data (provided animals, acquired and managed patients, induced lung cancer in vitro and in vivo (34). To our knowledge, provided facilities, etc.): Y. Zhang, R.G. Lapidus, P. Liu, E.Y. Choi, S. Adediran, this agent has not yet been evaluated in prostate cancer. In the X. Wang, H.C. Dan present study, we evaluated the anticancer activity of CmpdA in Analysis and interpretation of data (e.g., statistical analysis, biostatistics, vitro and in vivo in two prostate cancer cell lines that have activated computational analysis): Y. Zhang, P. Liu, E.Y. Choi, A. Hussain, X. Liu, H.C. Dan IKKb. CmpdA led to significant inhibition of cell proliferation, cell Writing, review, and/or revision of the manuscript: Y. Zhang, R.G. Lapidus, migration, and EMT transition, and was associated with enhanced A. Hussain, X. Liu, H.C. Dan apoptosis in prostate cancer cells. Furthermore, CmpdA signifi- Administrative, technical, or material support (i.e., reporting or organizing cantly inhibited the expression of transcription factors Nanog, data, constructing databases): E.Y. Choi, X. Liu, H.C. Dan Sox-2, and Oct-4 in both prostate cancer cells in cell culture and Study supervision: X. Liu, H.C. Dan xenograft tumors, suggesting its potential in suppression of prostate cancer stemness. Importantly, CmpdA, as single agent, caused Acknowledgments inhibition of tumor xenografts without significant loss of mouse The authors thank Dr. Albert Baldwin in University of North Carolina at body weight, suggesting that it may be associated with reduced Chapel Hill for providing the IKKb inhibitor CmpdA. toxicity in vivo. Given that targeting a single activated pathway is unlikely to provide meaningful long-term tumor control in a Grant Support complex disease state such as prostate cancer, we also evaluated This work was supported, in part, by NIH Grant R00CA149178 and startup the effects of combining docetaxel chemotherapy with CmpdA. funds from Marlene and Stewart Greenebaum Cancer Center, University of That significant synergism exists between docetaxel and CmpdA Maryland School of Medicine (to H.C. Dan) and a Merit Review Award, in inhibiting prostate cancer cells suggests that docetaxel, in Department of Veterans Affairs (to A. Hussain). The costs of publication of this article were defrayed in part by the payment of combination with IKKb targeting, may be a viable strategy to page charges. This article must therefore be hereby marked advertisement in explore further in future studies. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. In summary, we demonstrate that activated IKKb may represent a useful target, thus providing a rationale for exploring the Received December 21, 2015; revised March 14, 2016; accepted March 28, therapeutic effects of novel IKKb inhibitors with favorable toxicity 2016; published OnlineFirst April 8, 2016.

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www.aacrjournals.org Mol Cancer Ther; 2016 OF11

Targeting IκB Kinase β/NF-κB Signaling in Human Prostate Cancer by a Novel I κB Kinase β Inhibitor CmpdA

Yanting Zhang, Rena G. Lapidus, Peiyan Liu, et al.

Mol Cancer Ther Published OnlineFirst April 8, 2016.

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