Mechanistic Elucidation of the Antitumor Properties of Withaferin a in Breast Cancer

Published OnlineFirst April 14, 2014; DOI: 10.1158/0008-5472.CAN-13-2081

Cancer Tumor and Stem Cell Biology Research

Mechanistic Elucidation of the Antitumor Properties of Withaferin A in Breast Cancer

Arumugam Nagalingam1, Panjamurthy Kuppusamy2, Shivendra V. Singh3, Dipali Sharma1, and Neeraj K. Saxena2

Abstract Withaferin A (WFA) is a steroidal lactone with antitumor effects manifested at multiple levels that are mechanistically obscure. Using a phospho-kinase screening array, we discovered that WFA activated phosphorylation of the S6 kinase RSK (ribosomal S6 kinase) in breast cancer cells. Pursuing this observation, we deﬁned activation of extracellular signal–regulated kinase (ERK)–RSK and ETS-like transcription factor 1 (Elk1)–CHOP (C-EBP homologous protein) kinase pathways in upregulating transcription of the death receptor 5 (DR5). Through this route, WFA acted as an effective DR5 activator capable of potentiating the biologic effects of celecoxib, etoposide, and TRAIL. Accordingly, WFA treatment inhibited breast tumor formation in xenograft and mouse mammary tumor virus (MMTV)-neu mouse models in a manner associated with activation of the ERK/ RSK axis, DR5 upregulation, and elevated nuclear accumulation of Elk1 and CHOP. Together, our results offer mechanistic insight into how WFA inhibits breast tumor growth. Cancer Res; 74(9); 1–13. 2014 AACR.

Introduction somnifera, is composed of 14 bioactive compounds known as Breast cancer is the most commonly diagnosed cancer in withanolides (6, 7). Withaferin A (WFA) is the most abundant women worldwide. Despite improvements in early diagnosis, and therapeutically effective withanolide and multiple studies – and development of various targeted therapeutic approaches, have shown the anticancer activities of WFA (8 10). WFA breast cancer–related mortality still remains at a high level. administration decreases mammary tumors and pulmonary Major limitations associated with available therapeutic metastasis in the MMTV (mouse mammary tumor virus)-neu approaches are high toxicity, lower efficacy, therapeutic resis- transgenic model and is associated with increased apoptosis tance, and therapy-related morbidity. A more promising (11) WFA-induced apoptosis involves reactive oxygen species approach seems to be the development of more effective, production (12), FOXO3a and Bim induction (8). Although nonendocrine, nontoxic therapeutic strategies using active WFA effectively inhibits oncogenic transcription factors such constitutive agents in natural products owing to their cancer as Stat3 (13), resulting in growth inhibition, it is interesting to preventive as well as therapeutic potential. Historically, natural note that WFA has also been reported to activate Notch products have played an important role in the discovery and signaling, which plays an oncogenic role and is often hyper- development of novel anticancer agents (1, 2). Withania som- active in breast cancer cells (14). Multiple other targets have k nifera has been successfully used in traditional ayurvedic been documented for WFA action such as NF- B, BCL2, Hsp90, – medicine to treat various diseases owing to its broad spectrum vimentin, and annexin II in various other cellular systems (15 pharmacologic efficacy (3–5). The root extract of Withania 20); however, a complete molecular understanding of WFA- mediated signaling networks in breast cancer growth inhibition is still emerging. Deciphering the key nodes of WFA action Authors' Affiliations: 1Department of Oncology, Johns Hopkins University in breast cancer is needed to establish surrogate biomarkers School of Medicine and the Sidney Kimmel Comprehensive Cancer Center fi at Johns Hopkins; 2Department of Medicine, University of Maryland School for its ef cacy and help in clinical development of this bioactive of Medicine, Baltimore Maryland; and 3Department of Pharmacology and molecule, an issue, we addressed by systematically elucidating Chemical Biology, University of Pittsburgh Cancer Institute, University of the underlying mechanisms. Pittsburgh School of Medicine, Pittsburgh, Pennsylvania Because many cellular signaling events involve induced Note: Supplementary data for this article are available at Cancer Research phosphorylation of key targets, in the present study, we used Online (http://cancerres.aacrjournals.org/). phosphokinase arrays to gain insight into the intricacies of Corresponding Authors: Dipali Sharma, Department of Oncology and the Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University WFA-mediated signaling networks and discovered that WFA School of Medicine, 1650 Orleans Street, CRB 1, Room 145, Baltimore, MD induces ribosomal S6 kinase (RSK) phosphorylation in breast 21231. Phone: 410-455-1345; Fax: 410-614-4073; E-mail: cancer cells. We designed this study to examine the role of RSK [email protected]; and Neeraj K. Saxena, Department of Medicine, University of Maryland School of Medicine, 660 W Redwood Street, and the underlying molecular mechanisms how WFA-medi- Howard Hall, Room 301, Baltimore, MD 21201. E-mail: ated activation of RSK leads to growth inhibition of breast [email protected] cancer cells. Here, we provide strong evidence for extracellular doi: 10.1158/0008-5472.CAN-13-2081 signal–regulated kinase (ERK)/RSK as an important mediator 2014 American Association for Cancer Research. in WFA-induced breast cancer growth inhibition and uncover

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a novel mechanism of WFA action through activation of the expression of these proteins by Western blotting. In this ETS-like transcription factor 1 (Elk1)/C-EBP homologous pro- study, WFA administration resulted in a statistically signif- tein (CHOP) axis leading to death receptor 5 (DR5) activation. icant decrease in macroscopic mammary tumor size, micro- scopic mammary tumor area (11). All animal studies were in accordance with the guidelines of Johns Hopkins University Materials and Methods Institutional Animal Care and Use Committee (IACUC) and Cell culture and reagents University of Pittsburgh IACUC. The human breast cancer cell lines, MCF7, MDA-MB-231, T47D, and MDA-MB-468 were obtained from the American Phospho-antibody array analysis Type Culture Collection, resuscitated from early passage liquid Breast cancer cells were treated with WFA and the phospho- nitrogen vapor stocks as needed and cultured according to the antibody array analysis was performed using the Proteome supplier's instructions. Cells were cultured for less than 3 Profiler Human Phospho-Kinase Array Kit ARY003 from R&D months before reinitiating cultures and were routinely Systems according to the manufacturer's instructions. Array inspected microscopically for stable phenotype. WFA was images were analyzed using the GeneTools image analysis procured from Calbiochem. Fluoromethyl ketone–methox- software (Syngene). yethylamine (fmk–MEA), a specific p90-RSK inhibitor was kindly provided by Dr. Jack Taunton (University of California Subcellular fractions, immunoblotting, transfection, at San Francisco, Cellular and Molecular Pharmacology, San RNA interference, immunofluorescence, and confocal Francisco, CA; ref. 21). U0126 and trichostatin A (TSA) were imaging procured from Sigma. CHOP overexpression construct was Cellular cytosolic and nuclear fractions were prepared fol- purchased from OriGene Technologies, Inc. Wild-type and lowing previously published protocol (25). Immunoblotting S383A-mutant Elk1 plasmids were kindly provided by Dr. was carried out as described (26). The blots are representative Andrew D. Sharrocks (University of Manchester, Manchester, of multiple independent experiments and bar diagrams are UK; ref. 22). ERK plasmids were kindly provided by Dr. Paul included showing quantitation of Western blot signals. Breast Shapiro, University of Maryland (Baltimore, MD). Celecoxib cancer cells were transfected with ERK, CHOP, Elk1-WT, and was obtained from LKT laboratories. TRAIL was obtained from Elk1-S383A–mutant vectors using Lipofectamine-2000 (Invi- Millipore. Etoposide was purchased from Sigma. trogen) and treated with WFA as indicated. For RNA interference, cells were transfected at 50% confluency with 100 nmol/L Clonogenicity, anchorage-independent growth, and cell of control siRNA or ERK1/2 siRNA (SignalSilence) using Oli- viability assays gofectamine (Cell Signaling Technology). Breast cancer cells To perform clonogenicity assay (23), breast cancer cells were were subjected to immunofluorescence analysis as described treated with WFA as indicated for 10 days; colonies were previously (24). counted. Anchorage-independent growth of breast cancer cells in the presence of WFA was assayed by colony formation in soft Chromatin immunoprecipitation and RNA isolation, agar (24). Cell viability assay was performed using a commer- RT-PCR cially available XTT Assay Kit (Roche Applied Science). Chromatin immunoprecipitation (ChIP) analyses were performed using our published procedure (27). Total cellu- Breast tumorigenesis assay lar RNA was extracted using the TRIzol Reagent Kit (Life MDA-MB-231, MDA-MB-231-pLKO.1, MDA-MB-231- Technologies, Inc.). RT-PCR was performed using specific DR5shRNA1, and MDA-MB-231-DR5shRNA2 xenografts were sense and antisense PCR primers. generated as previously described (24), grouped in two experimental groups (8 mice/group) and treated with intra- Stable knockdown using lentiviral short hairpin RNA peritoneal injections of either vehicle (10% dimethyl sulfox- Five to six premade lentiviral DR5, CHOP and RSK short ide, 40% cremophor-EL, and 50% PBS) or vehicle containing hairpin RNA (shRNA) constructs, and a negative control con- 4-mg WFA (ChromaDex Inc.)/kg body weight 5 d/wk for struct created in the same vector system (pLKO.1) were 5 weeks. The dose and route of WFA administration were purchased from Open Biosystems. Constructs were used for selected from previous study documenting in vivo efficacy of transient transfections using Fugene or Lipofectamine. Paired WFA (8). Tumors were collected after 4 weeks of treatment; stable knockdown cells were generated following our previ- measured, weighed, and subjected to further analysis by ously established protocol (25). immunohistochemistry, real-time PCR (RT-PCR), and West- ern blotting. At least four random, nonoverlapping repre- Statistical analysis sentative images from each tumor section from eight tumors All experiments were performed thrice in triplicates. of each group were captured using ImagePro software for Statistical analysis was performed using Microsoft Excel quantitation of pERK (phosphorylated-ERK), pRSK (phos- software. Significant differences were analyzed using the phorylated-RSK), CHOP, pElk1 (phosphorylated-Elk1), and Student t test and two-tailed distribution. Results were DR5 expression. MMTV-neu mice model–mammary tumor considered to be statistically significant if P < 0.05. Results tissues from our previously published prevention study in were expressed as mean SE between triplicate experi- MMTV-neu mice (11) were also used to determine the ments performed thrice.

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Withaferin A Modulates ERK/RSK and CHOP/Elk1 Axes in Breast Cancer

Results with WFA (Supplementary Fig. S2A). An induction of cleaved WFA treatment inhibits clonogenicity, anchorage- PARP was observed in the presence of 5 and 10 mmol/L WFA independent growth of breast cancer cells and inhibits (Supplementary Fig. S2A). Treatment of breast cancer cells breast tumor progression in athymic nude mice with 5 mmol/L WFA for various time intervals showed a We first examined the effect of WFA on clonogenic potential decrease in XIAP expression along with an induction of PARP and anchorage-independent growth of breast cancer cells. cleavage (Supplementary Fig. S2B). Treatment with 5 mmol/L WFA resulted in approximately We further investigated the in vivo physiologic relevance of 50% to 60% inhibition in clonogenicity and soft agar colony our in vitro findings by evaluating whether WFA had inhibitory formation, whereas higher concentrations were more inhibi- effects on the development of breast carcinoma in nude mouse tory (Fig. 1A and B). Exposure of breast cancer cells to WFA led models. Tumor growth was significantly inhibited in the WFA- to decreased cell viability (Supplementary Fig. S1A–S1D). treated experimental group in comparison with the control WFA-mediated inhibition of cancer cell growth is associated group (Fig. 1C). Ki-67, a nuclear nonhistone protein, is one of with induction of apoptosis (8, 12, 17). Members of inhibitor-of- the major markers of tumor proliferation (31) used as a apoptosis protein family, survivin and X-linked inhibitor-of- decision-making tool for adjuvant therapy (32). The immuno- apoptosis protein (XIAP), mainly function to suppress apo- histochemical assessment of tumor proliferation showed ptosis by inhibiting caspases (28) and are associated with higher Ki-67 in the control group as compared with the increased aggressiveness, higher recurrence rate, and unfavor- WFA-treated group (Fig. 1D and E). In our in vitro analysis, able breast cancer outcome (28–30). Decreased expression of we found that WFA modulated the expression of survivin and survivin and XIAP was observed in breast cancer cells treated XIAP in breast cancer cells. Corroborating the in vitro findings,

Figure 1. WFA inhibits clonogenicity, anchorage-independent growth and breast tumor growth in nude mice. A, clonogenicity of breast cancer cells treated with various concentrations of WFA (as indicated). B, soft agar colony formation of breast cancer cells treated with WFA for 3 weeks. Histogram represents average number of colonies counted (in six microﬁelds). , P < 0.001, compared with untreated controls. C, vehicle-treated cells. C, MDA- MB-231 cell–derived tumors were developed in nude mice and treated with vehicle or WFA. Tumor growth was monitored by measuring the tumor volume for 5 weeks (n ¼ 8 mice/group; P < 0.001). D and E, tumors from vehicle (V) and WFA-treated mice were subjected to immunohistochemical analysis using Ki-67, survivin, and XIAP antibodies. Bar diagrams, quantitation of immunohistochemical analysis. Columns, mean (n ¼ 8); bar, SD. , signiﬁcantly different (P < 0.005) compared with control. F, TUNEL-positive cells in tumor sections were counted. Bars, the mean (n ¼ 6–8). , P < 0.01, compared with untreated controls.

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Figure 2. Human phospho- antibody array analyses reveal WFA-induced increased phosphorylation of RSK and ERK and WFA activates RSK in an ERK- dependent manner. A and B, MCF7 and MDA-MB-231 breast cancer cells were treated with 5 mmol/L WFA for 3 hours and subjected to human phospho-antibody array analyses. Relative levels of protein phosphorylation (normalized intensity for each antibody) were calculated for each untreated and treated sample. , P < 0.001, compared with untreated controls. C, immunoblot analysis of pRSK– Ser380 and total RSK in breast cancer cells treated with WFA as indicated. D, breast cancer cells were treated with 5 mmol/L WFA for indicated time intervals. Total lysates were immunoblotted for pRSK and total RSK expression. E, breast cancer cells were treated as in C; lysates were examined for pERK44/42 and total ERK. F, breast cancer cells were treated as in D; total lysates were immunoblotted for pERK and total ERK expression. G, breast cancer cells were transiently transfected with siERK–siRNAs for 48 hours followed by immunoblot analysis of ERK levels. H, breast cancer cells were transfected with siERK as in G, followed by WFA treatment (5 mmol/L, 3 hours) and immunoblot analysis of pRSK and total RSK levels. C, vehicle-treated cells.

the tumors from the WFA-treated mice exhibited lower expres- To identify cellular signal transduction pathways involved in sion of survivin and XIAP (Fig. 1D and E). The number of WFA-induced inhibition of breast carcinogenesis, we interro- TUNEL-positive apoptotic cells was increased in tumors from gated 46 speciﬁc Ser/Thr/Tyr phosphorylation sites of 38 the WFA-treated mice compared with the vehicle control selected proteins using phosphoprotein arrays. Breast cancer group (Fig. 1F). Collectively, these results show that WFA cells were treated with 5 mmol/L WFA for 3 hours and sub- treatment results in suppression of tumor growth, inhibition jected to phospho-protein analysis. Phosphorylation level of of cellular proliferation, and increased apoptosis in the breast p90-RSK was signiﬁcantly increased in both MCF7 and MDA- tumors. MB-231 cells. In addition, WFA treatment increased phosphorylation of ERK in breast cancer cells (Fig. 2A and B and WFA-dependent changes in phosphorylation of signaling Supplementary Fig. S3). Dysregulated RSK expression or activ- mediators in breast cancer cells ity has been associated with several human cancers, control- Phosphorylation of kinases is fundamentally important to ling various downstream signaling pathways and modulating multiple aspects of signaling networks and cellular functions. cellular processes (33, 34). In contrast with the conventional

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Withaferin A Modulates ERK/RSK and CHOP/Elk1 Axes in Breast Cancer

oncogenic role of RSK, multiple studies have shown RSK as a important role in mediating biologic effects of WFA in breast potential tumor suppressor, a participant in p53-dependent cancer cells. cell growth arrest, showing an important role in decreasing cancer cell proliferation, invasion, and metastasis (33–35). We WFA induces concomitant upregulation and nuclear further explored the molecular mechanisms how WFA acti- translocation of Elk1 and CHOP via the ERK/RSK vates RSK and the biologic significance of RSK activation in signaling axis in breast cancer cells WFA-mediated inhibition of breast carcinogenesis. The ETS domain transactivation factor Elk1 is a direct target of the MAPK pathways and phosphorylation of the Elk1 WFA treatment activates p90-RSK via ERK-dependent transcriptional activation domain by MAPKs triggers its acti- manner in breast cancer cells vation, which further induces apoptosis and growth inhibition To confirm whether WFA can activate RSK as indicated by (37). Phosphorylation at serine 383 plays a critical role in Elk1 phosphoprotein array results, and to clarify its dose- and time- activation. Increased Elk1 phosphorylation was observed with- dependent response, we treated breast cancer cells with WFA. in 30 minutes of WFA treatment, which was diminished after 6 Immunoblot analysis showed that phosphorylation of RSK hours posttreatment (Fig. 3A and B). We examined whether increased in a concentration-dependent manner in both MCF7 WFA-induced Elk1 phosphorylation involves ERK. Indeed, and MDA-MB-231 cells (Fig. 2C). Treatment of breast cancer pretreatment of breast cancer cells with ERK siRNA reduced cells with 5 mmol/L WFA for various time intervals exhibited WFA-induced phosphorylation of Elk1, indicating a direct striking increase in RSK phosphorylation within 30 minutes regulatory role of ERK (Supplementary Fig. S7A and S7B). Our (Fig. 2D and Supplementary Fig. S4A), whereas total RSK studies discovered the involvement of RSK in WFA function, remained unchanged. RSK activation is controlled by the therefore, we investigated whether RSK is integral for WFA- canonical Ras/mitogen-activated protein kinase (MAPK) path- mediated Elk1 phosphorylation. RSK-specific inhibitor fmk– way via direct phosphorylation of RSK by ERK (34). ERK itself MEA inhibited phosphorylation of RSK (Fig. 3C). Pretreatment represents a major survival signaling pathway that promotes of breast cancer cells with fmk–MEA followed by WFA treat- cancer cell survival via inhibiting apoptosis; however, a proa- ment showed that inhibition of RSK phosphorylation inhibited poptotic role of ERK signaling has also been shown (36). Our WFA-induced Elk1 phosphorylation (Fig. 3D and Supplemen- phosphoprotein array analysis showed elevated phosphoryla- tary Fig. S7C). Phosphorylation can also affect the subcellular tion of ERK in breast cancer cells treated with WFA. Because of localization of Ets proteins (37, 38). As evident in Fig. 3E, WFA these interesting attributes, we explored the effect of WFA on treatment did not affect the total expression level of Elk1 in ERK activation. Increased phosphorylation of ERK was either cytoplasmic or nuclear fraction; however, increased level observed in response to WFA treatment as compared with of phosphorylated Elk1 was observed in nuclear fraction within untreated cells (Fig. 2E). Breast cancer cells, treated with 5 30 minutes after treatment, which remained elevated for mmol/L WFA for various intervals of time, showed that WFA the duration of the treatment (Fig. 3E and Supplementary increased ERK phosphorylation within 30 minutes of WFA Fig. S7D). Immunofluorescence analysis of breast cancer cells treatment (Fig. 2F and Supplementary Fig. S4B). We ques- showed nuclear phosphorylated Elk1 in WFA-treated cells tioned whether ERK is a key player in WFA-mediated RSK (Fig. 3F). activation and toward this end, we used ERK siRNA to silence Induction of apoptotic response by many pharmacologically ERK (Fig. 2G) and then investigated its impact on WFA- active compounds and anticancer drugs is achieved by acti- induced RSK activation. Immunoblot analysis for phosphory- vation of endoplasmic reticulum (ER) stress. ER stress signal- lated RSK levels after ERK silencing and WFA treatment ing system induces transcription of CHOP, a key proapoptotic showed that indeed ERK silencing inhibited WFA-induced transcription factor that binds with other transcription factors RSK phosphorylation (Fig. 2H and Supplementary Fig. S4C). and induces proapoptotic genes (39). We found that WFA Inhibition of ERK phosphorylation using U0126 also resulted in increased expression of CHOP in a temporal manner with a abrogation of WFA-mediated RSK activation (Supplementary significant increase observed within 30 minutes after treat- Fig. S5A). Next, we examined the importance of ERK and RSK ment (Fig. 4A and B). To evaluate the contribution of ERK in activation in WFA-mediated apoptosis in breast cancer cells WFA-induced CHOP overexpression, we examined CHOP using MEK/ERK inhibitor, U0126, and RSK inhibitor, fmk– expression after ERK silencing using ERK siRNA. Results MEA. WFA treatment resulted in elevated PARP cleavage, indicated that indeed ERK silencing abrogated WFA-induced indicating increased apoptotic response in breast cancer cells, CHOP expression (Fig. 4C and D), suggesting the involvement which was inhibited in breast cancer cells cotreated with of ERK in the CHOP-stimulatory effect of WFA. Once upregu- U0126 or fmk–MEA (Supplementary Fig. S5B and S5C). ERK lated, CHOP translocates to the nucleus and participates in silencing abrogated WFA-mediated inhibition of clonogenicity transcriptional modulation of responsive genes critical for of breast cancer cells and reintroduction of ERK in ERK- tumor cell apoptosis (39). Analysis of nuclear and cytoplasmic silenced cells resensitized them to WFA (Supplementary Fig. fractions from WFA-treated cells showed that WFA treatment S6A). RSK inhibition using RSK–shRNA(s) (Supplementary Fig. increased nuclear accumulation of CHOP (Fig. 4E and F). S6B) also rendered breast cancer cells nonresponsive to WFA- Immunostaining of MCF7 and MDA-MB-231 cells showed mediated inhibition of clonogenicity of breast cancer cells that WFA treatment increases nuclear accumulation of (Supplementary Fig. S6C). Collectively, these pieces of evidence CHOP, whereas untreated cells showed predominantly cyto- support the notion that RSK and ERK activation plays an plasmic localization of CHOP (Fig. 4G). CHOP inhibition using

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Figure 3. The RSK signaling axis mediates WFA-induced phosphorylation of Elk1. A, immunoblot analysis of pElk1 and Elk1 in breast cancer cells treated with 5 mmol/L WFA for indicated time intervals. B, bar diagram, quantitation of Western blot signals from multiple independent experiments. , P < 0.005, compared with untreated controls. C, breast cancer cells were treated with RSK inhibitor fmk–MEA (6 mmol/L) for indicated time intervals. Total lysates were immunoblotted for pRSK and total RSK expression. D, breast cancer cells were pretreated with fmk–MEA (6 mmol/L, 3 hours) followed by WFA treatment (5 mmol/L, 3 hours) as indicated. Total lysates were immunoblotted for pElk1 and total Elk1 expression. E, immunoblot analysis of pELK1 and total Elk1 in cytoplasmic and nuclear fractions of breast cancer cells treated with 5 mmol/L WFA for indicated time intervals. F, breast cancer cells were treated with 5 mmol/L WFA for 6 and 24 hours as indicated and subjected to immunoﬂuorescence analysis of pElk1. Nuclei were visualized with 40,6-diamidino-2- phenylindole (DAPI) staining. C, vehicle-treated cells.

CHOP–shRNA(s) (Supplementary Fig. S8A) rendered breast drugs and bioactive molecules (40, 41). We found that WFA cancer cells nonresponsive to WFA-mediated inhibition of induced the expression of DR5 in breast cancer cells (Fig. 5A and clonogenicity of breast cancer cells (Supplementary Fig. Supplementary Fig. S9A–S9D). The fact that DR5 promoter S8B), exhibiting importance of CHOP in WFA function. These contains cis-acting CHOP-like binding sequence prompted us ﬁndings suggest that the ERK/RSK signaling axis plays an to explore the connection between WFA-mediated CHOP important role in regulating upregulation and activation of induction and DR5 upregulation. We investigated whether CHOP and Elk1 in breast cancer cells. enforced overexpression of CHOP (Fig. 5B) using CHOP overexpression construct alters WFA-mediated DR5 expression. Elk1 and CHOP contribute to WFA-induced upregulation Overexpression of CHOP in MCF7 and MDA-MB-231 cells of DR5 in breast cancer cells increased DR5 expression potentiating the effect of WFA (Fig. DR5 is a death domain–containing transmembrane receptor 5C and Supplementary Fig. S9E). In a reciprocal approach, that triggers apoptotic response upon overexpression or acti- CHOP was silenced using CHOP–shRNA approach (Supplemen- vation by certain stimuli, including small-molecule anticancer tary Fig. S8A) followed by WFA treatment. Silencing of CHOP in

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Figure 4. WFA increases CHOP expression via ERK and promotes its nuclear translocation. A, immunoblot analysis of CHOP in breast cancer cells treated with 5 mmol/L WFA for indicated time intervals. B, bar diagram, quantitation of Western blot signals from multiple independent experiments. , P < 0.001, compared with untreated controls. C, breast cancer cells were transfected with siERK for 48 hours followed by WFA (5 mmol/L, 3 hours) treatment and immunoblot analysis using anti-CHOP antibodies. C, control-transfected cells. D, bar diagram, quantitation of Western blot signals from multiple independent experiments. , P < 0.001, compared with untreated controls; , P < 0.05, compared with WFA-treated cells. E, immunoblot analysis of CHOP in cytoplasmic and nuclear fractions of breast cancer cells treated with 5 mmol/L WFA for indicated time intervals. F, bar diagram, quantitation of Western blot signals from multiple independent experiments. , P < 0.05, compared with untreated controls. G, breast cancer cells were treated with 5 mmol/l WFA for 6 and 24 hours and subjected to immunoﬂuorescence analysis of CHOP. Nuclei were visualized with DAPI staining. C, vehicle-treated cells.

breast cancer cells inhibited WFA-mediated induction of DR5 phoacceptor residue Ser383. Breast cancer cells were trans- expression (Fig. 5D). A recent study reported that DR5 promoter fected with wild-type or phospho-deficient (S383A) Elk1 con- contains a putative Elk1-binding site and mutations in this structs. Expression of wild-type Elk1 increased DR5 expression, binding site affected DR5 transactivation, suggesting a link whereas phospho-deficient Elk1 did not affect DR5 expression between DR5 and Elk1 (42). Because there was no previous (Fig. 5E). To examine whether Elk1 contributes to WFA-medi- study linking Elk1 to DR5 expression in breast cancer cells, we ated DR5 upregulation, we transfected breast cancer cells with asked whether Elk1 was indeed involved in regulating WFA- wild-type or phospho-deficient Elk1 constructs followed by mediated DR5 expression. Transactivation potential of Elk1 is WFA treatment. Wild-type Elk1 potentiated, whereas phos- enhanced by S383 phosphorylation (38), which is increased by pho-deficient Elk1 inhibited WFA-mediated DR5 expression WFA (Fig. 3A and B); therefore, we focused on the key phos- (Fig. 5F and Supplementary Fig. S9F) demonstrating the

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Figure 5. CHOP and Elk1 contribute to WFA-induced upregulation of DR5 in breast cancer cells. A, immunoblot analysis of DR5 in breast cancer cells treated with 5 mmol/L WFA for indicated time intervals. B, breast cancer cells were transfected with CHOP overexpression plasmid (CHOPO/E)and analyzed for CHOP expression using immunoblot analysis. C, immunoblot analysis of CHOP and DR5 in breast cancer cells transfected with CHOPO/E followed by WFA (5 mmol/L, 3 hours) treatment (CHOPO/E þ WFA). D, breast cancer cells were transfected with CHOP-shRNA1 followed by WFA (5 mmol/l, 3 hours) treatment. Total lysates were analyzed for DR5 expression. E, breast cancer cells were transfected with Elk1 wild-type (Elk1 WT) and S383A phospho-mutant Elk1 (Elk1M) plasmids. Total lysates were analyzed for pElk1, total Elk1, and DR5 expression. F, MCF7 cells were transfected with Elk1 WT and Elk1M as in E, followed by WFA (5 mmol/L, 3 hours) treatment. C, untransfected controls. Total lysates were immunoblotted for pElk1, total Elk1, and DR5 expression. G, immunoblot analysis of pRSK, total RSK, and DR5 in MDA-MB-231 cells pretreated with fmk–MEA (6 mmol/L, 3 hours) followed by WFA (5 mmol/L, 3 hours) treatment. H, breast cancer cells were transfected with RSK–shRNA1 followed by WFA (5 mmol/L, 3 hours) treatment. Total lysates were analyzed for DR5 expression. I, MCF7 cells were treated with 5 mmol/l WFA for 3 and 6 hours and subjected to ChIP assay using CHOP, pElk1, HDAC1, and acetylated-H4 antibodies. The puriﬁed DNA was analyzed by PCR using speciﬁc primers spanning the CHOP and Elk1-binding sites on DR5 promoter. C, vehicle-treated cells.

important role of p-Ser383-Elk1 in WFA action. Our studies also could modulate WFA-mediated DR5 upregulation. Indeed, showed an important role of RSK phosphorylation in WFA- inhibition of RSK phosphorylation resulted in inhibition of mediated Elk1 phosphorylation (Fig. 3D and Supplementary Fig. WFA-induced DR5 expression (Fig. 5G). RSK was silenced using S7C); therefore, we examined whether RSK inhibitor fmk–MEA RSK–shRNA approach (Supplementary Fig. S6B) followed by

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Withaferin A Modulates ERK/RSK and CHOP/Elk1 Axes in Breast Cancer

WFA treatment. Silencing of RSK in breast cancer cells inhibited cinoma in nude mouse models. MDA-MB-231-pLKO.1 and WFA-mediated induction of DR5 expression (Fig. 5H). In an MDA-MB-231-DR5shRNA1 and shRNA2 were used in the xenograft effort to better understand the molecular events involved in athymic nude mice model. Tumor growth was significantly WFA-induced DR5 expression, we used ChIP analyses to exam- inhibited in WFA-treated MDA-MB-231-pLKO.1 (the vector ine the recruitment of pElk1 and CHOP to the DR5 promoter. control group), whereas WFA was unable to inhibit tumor pElk1 and CHOP were associated with the DR5 promoter in the growth in MDA-MB-231-DR5shRNA1 and shRNA2 groups (Fig. 6J– presence of WFA. DR5 in repressed state has been shown to be L). These results showed that WFA-induced DR5 overexpres- associated with HDAC1 (43). Intriguingly, we observed release sion is indeed a crucial component of the signaling machinery of HDAC1 from DR5 promoter in response to WFA treatment used by WFA in inhibiting growth of breast cancer cells. and a significant increase in histone H4 acetylation, indicating active chromatin conformation (Fig. 5I and Supplementary Fig. WFA administration modulates activation of ERK/RSK S10A). Treatment of breast cancer cells with HDAC inhibitor, and CHOP/Elk1 axes in vivo TSA, resulted in increased expression of DR5 (Supplementary Our studies show that WFA treatment inhibits breast tumor Fig. S10B) supporting a functional role of HDAC1 in regulation progression in vivo (Fig. 1C). We used tumor tissue from the of DR5 expression. Taken together, these data show that WFA same experiment to determine the effect of WFA treatment on induces DR5 expression in breast cancer cells through a mech- the expression and activation of important signaling mole- anism involving CHOP and Elk1. cules. Tumors from WFA-treated mice exhibited increased phosphorylation of ERK, RSK, Elk1 as well as higher expression WFA is a potent inducer of DR5 and silencing of DR5 CHOP and DR5 in comparison with the vehicle-treated group abrogates WFA-mediated growth inhibition of breast (Fig. 7A). Also, WFA-treated tumors showed transcriptional cancer cells upregulation of DR5 (Fig. 7B). Immunohistochemical analysis DR5, upon induction, mediates enhancement of TRAIL- showed that tumors from WFA-treated mice exhibited higher induced apoptosis and contributes to apoptosis by other number of tumor cells showing increased expression of pERK, small-molecule drugs (44). Using established small-molecule pRSK, CHOP, pELK1, and DR5 as compared with tumors from DR5 inducers (TRAIL, celecoxib, and etoposide), we com- the vehicle-treated group (Fig. 7C and D) providing physiologic pared the efficacy of WFA to induce DR5 expression in relevance to our in vitro findings. Earlier studies from our group breast cancer cells. It was interesting to note that WFA have shown that WFA treatment prevents mammary cancer treatment resulted in a greater induction of DR5 expression development in the MMTV-neu mouse model, tumor weight in in comparison with TRAIL, celecoxib, and etoposide (Fig. 6A the WFA treatment group was lower by 50% in comparison and Supplementary Fig. S11A). To determine whether WFA with the control group (11). Mammary tumor tissues from this enhances the effect of small-molecule DR5 inducers, we study were used to examine the ERK/RSK signaling axis and treated breast cancer cells with WFA in combination with DR5. Tumors from the WFA-treated group exhibited increased TRAIL, etoposide, and celecoxib and assessed modulation of expression of phosphorylated ERK and RSK as well as higher DR5 expression, clonogenicity, and soft agar colony forma- expression of DR5 in comparison with the control group (Fig. tion. Combination treatment with WFA enhanced etopo- 7E). Collectively, the findings presented here suggest that WFA side-, celecoxib-, and TRAIL-induced DR5 expression (Fig. 6B inhibits breast tumor progression and provide in vitro as well as and Supplementary Fig. S11B) and resulted in significantly in vivo evidence for the involvement of RSK as an important higher inhibition of clonogenicity and soft agar colony mediator, and uncover a novel mechanism of WFA action formation (Fig. 6C and D). These results show that WFA is through activation of Elk1 leading to DR5 activation. apotentinducerofDR5thatcanactasaneffectivebioactive alternative to TRAIL, celecoxib, and etoposide as well as enhance their effect when used in combination. Discussion To directly examine the role of DR5 in WFA-mediated The realization of the ability of bioactive small-molecule growth inhibition of breast cancer cells, we used DR5shRNA agent WFA to effectively inhibit carcinogenesis in a nontoxic, lentiviruses and puromycin to select for stable pools of MCF7 nonendocrine manner has made this agent of potential interest and MDA-MB-231 cells with DR5 depletion. We analyzed in the treatment of breast cancer and sparked a new interest in pLKO.1 and DR5shRNA stable MCF7 and MDA-MB-231 cell understanding the underlying molecular mechanisms. Deci- pools for DR5 protein expression, and found that DR5 protein phering the key nodes of WFA action can help in establishing – expression was significantly knocked down in DR5shRNA1 3 surrogate biomarkers for its efficacy and help in clinical devel- cells as compared with pLKO.1 control cells (Fig. 6E). WFA opment of this bioactive molecule. We found that WFA effec- increased PARP cleavage in pLKO.1 cells, whereas no change in tively inhibits the growth of breast cancer cells in vitro and in cleaved PARP was observed in DR5shRNA MCF7 and DR5shRNA vivo but our phosphokinase array studies led us to novel MDA-MB-231 cells (Fig. 6F). WFA treatment efficiently inhib- discoveries that extend beyond the popular proliferative and ited clonogenicity and soft agar colony formation of pLKO.1 oncogenic role of certain kinases and inhibition of these kinases breast cancer cells but not of DR5shRNA cells (Fig. 6G–I). We by anticancer agents to achieve tumor growth inhibition. Both further investigated the in vivo physiologic relevance of our in p90-RSK and ERK are popularly known for their oncogenic role vitro findings by evaluating whether DR5 is integral for the but this study illustrates that WFA despite efficiently inhibiting inhibitory effects of WFA on the development of breast car- breast tumor growth, increases phosphorylation of RSK in

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Figure 6. WFA is a potent inducer of DR5 in breast cancer cells and stable knockdown of DR5 abrogates WFA-mediated inhibition of growth of breast cancer cells. A, immunoblot analysis of DR5 in MCF7 cells treated with 50 mmol/L celecoxib (CCB), 1 mmol/L etoposide (E), 100 ng/mL TRAIL (T), and 5 mmol/L WFA for 6 hours. B, MCF7 cells were treated with celecoxib, etoposide, TRAIL alone as in A or in combination with 5 mmol/L WFA for 6 hours. Cell lysates were immunoblotted for DR5 expression. C, breast cancer cells were treated as in B and subjected to clonogenicity assay. D, breast cancer cells were treated as in B and subjected to soft agar colony formation assay for 3 weeks. Results are expressed as average number of colonies counted (in six microﬁelds). , P < 0.001, compared with untreated controls; #, P < 0.001, compared with etoposide-treated cells; , P < 0.005, compared with celecoxib- treated cells; and , P < 0.001, compared with TRAIL-treated cells. C, vehicle-treated cells. E, immunoblot analysis of DR5 in stable pools of DR5- depleted (DR5 shRNA1-3) and vector control (pLKO.1) MCF7 and MDA-MB-231 cells. F, immunoblot analysis of cleaved PARP and total PARP in MCF7- DR5shRNA1, MCF7-pLKO.1, MDA-MB-231-DR5shRNA1, and MDA-MB-231-pLKO.1 cells treated with 5 mmol/L WFA. G, clonogenicity of MDA-MB-231- DR5shRNA1, MDA-MB-231-pLKO.1, MCF7-DR5shRNA1,3, and MCF7-pLKO.1 cells in the presence of WFA (5 mmol/L). H and I, soft agar colony formation in MDA-MB-231-DR5shRNA1, MDA-MB-231-pLKO.1, MCF7-DR5shRNA1,3, and MCF7-pLKO.1 cells in the presence of WFA (5 mmol/L). Results are expressed as average number of colonies counted (in six microﬁelds). , P < 0.001, compared with untreated pLKO.1 cells; #, P < 0.005, compared with untreated DR5shRNA cells. J–L, MDA-MB-231-pLKO.1, MDA-MB-231-DR5shRNA1, and MDA-MB-231-DR5shRNA2 cell–derived tumors were developed in nude mice and treated with vehicle or WFA. Tumor growth was monitored by measuring the tumor volume for 5 weeks (n ¼ 8 mice/group; P < 0.001). Representative tumor images are shown here. Tumor weight is shown in the table.

breast cancer cells via activation of ERK. These studies provide these data, we provide a schematic diagram depicting a series of an interesting mechanism by which WFA-induced ERK/RSK, in events, including a feed-forward interaction of ERK and RSK, a concerted action, results in concomitant upregulation/acti- direct involvement of CHOP/Elk1/HDAC1 in DR5 upregulation, vation of CHOP and Elk1. We also identify WFA as a potent DR5 which is operative in WFA-induced DR5-dependent growth activator in breast cancer whose effects mimic molecular and inhibition of breast cancer cells (Fig. 7F). cell biological outcomes of ligand-dependent DR5 activation. Our studies offer the ﬁrst evidence of the ability of WFA to Mechanistically, this process is associated with recruitment of activate RSK. RSK directly phosphorylates transcription fac- CHOP and Elk1 to DR5 promoter, increased histone acetylation tors, hence, regulating the transactivation of multiple gene in conjunction with clearance of histone deacetylase HDAC1. In targets. RSK positively regulates diverse cellular processes, vivo analyses of tumor xenografts and tumors from MMTV-neu including transformation, cell cycle, cell proliferation, cell mice provide further evidence of involvement of ERK/RSK and survival, and migration in response to various growth factors, CHOP/Elk1 axes and an integral role of DR5. On the basis of chemokines and other stimuli (45). RSK has been implicated in

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Withaferin A Modulates ERK/RSK and CHOP/Elk1 Axes in Breast Cancer

Figure 7. In vivo evidence for WFA- mediated activation of ERK/RSK and CHOP/Elk1 axes. A, Western blot analysis of indicated protein levels in MDA-MB-231 cell–derived tumors developed in nude mice and treated with vehicle or WFA for 5 weeks. B, total RNA was isolated from tumor samples and expression of DR5 was analyzed using RT-PCR analysis. C and D, tumors were subjected to immunohistochemical analysis using pERK, pRSK, CHOP, pELK1, and DR5 antibodies. Bar diagrams, quantitation of protein expression in tumors from vehicle and WFA- treated mice. Columns, mean (n ¼ 8); bar, SD. , signiﬁcantly different (P < 0.005) compared with control. E, tumor lysates from MMTV-neu mice treated with vehicle or WFA were immunoblotted for indicated proteins. F, schematic model of WFA-stimulated ERK/RSK signaling activating CHOP/Elk1, leading to DR5 induction and growth inhibition of breast cancer.

apoptosis inhibition achieved in part by phosphorylation of normal mesangial cells; ref. 41). Here, we show that WFA is Bad, CEBPb and DAPK (34), as well as apoptosis induction via more effective than three other known DR5 activators, phosphorylating Nur77 (46). We show that the ability of RSK to TRAIL, etoposide, and celecoxib, in activation of DR5 in mediate activation of Elk1 and CHOP leading to upregulation breast cancer cells, hence, seems to be a good candidate for of DR5 is critical to WFA outcomes in breast cancer cells. This further development as an effective DR5 inducer. It is inter- provides a new mechanism by which WFA can achieve effective esting to note that in recent years, many phase I and II single breast tumor growth inhibition. Also, these discoveries have agent and combination clinical trials have been conducted to potentially important implications in regard to increasing examine the efﬁcacy and safety of recombinant human interest in targeting breast cancer through manipulation of TRAIL (dulanermin; Amgen/Genentech), and the agonistic DR5. monoclonal antibodies to DR5 [lexatumumab (Human Activation of death receptors to induce apoptosis in tumor Genome Sciences), conatumumab (Amgen), drozitumab cells has been recognized as an effective way to therapeuti- (Genentech), tigatuzumab (Daiichi-Sankyo), and LBY135 cally target epithelial cell–derived cancers but this notion is (Novartis); refs. 44, 47]. Studies with these proapoptotic marred by the toxic effects of death receptor ligands, FASL receptor agonists (PARA) have been encouraging but no full and TNF. Over the few years, several small-molecule activa- phase III studies have been performed, suggesting the need tors of the death receptor family have been reported with for novel PARAs with improved properties. some more potent than others (44). WFA has been shown to Moreover, we report a novel ﬁnding that ERK activation is sensitize TRAIL-induced apoptosis and activates DR5 expres- important for WFA action in breast cancer cells. Generally sion in human renal cancer cells (Caki cells, but not in human considered a survival signaling pathway, ERK-mediated

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induction of apoptotic pathways has also been reported may potentially open new avenues of research on the role of (36, 48). Kinetics and duration of ERK activation are important WFA as a novel PARA. for these unusual effects of ERK. A transient activation of ERK inhibits cell death (49), whereas prolonged ERK activation is Disclosure of Potential Conflicts of Interest associated with the proapoptotic effect of ERK (36). WFA No potential conflicts of interest were disclosed. induces a sustained ERK activation in breast cancer cells, which was observed for the duration of experiment (until 24 Authors' Contributions Conception and design: A. Nagalingam, D. Sharma, N.K. Saxena hours). Very recently, it was reported that WFA increases ERK Development of methodology: A. Nagalingam, N.K. Saxena phosphorylation in MCF7 and SUM159 cells (50). It is known Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A. Nagalingam, S.V. Singh, N.K. Saxena that the ERK protein directly phosphorylates Elk1 (38). Hence, Analysis and interpretation of data (e.g., statistical analysis, biosta- the finding of the involvement of ERK in regulation of WFA- tistics, computational analysis): A. Nagalingam, P. Kuppusamy, D. Sharma, induced cell death resulted in our subsequent novel finding N.K. Saxena Writing, review, and/or revision of the manuscript: A. Nagalingam, that WFA activates Elk1, which contributes to DR5 induction. S.V. Singh, D. Sharma, N.K. Saxena In our study, inhibition of ERK, RSK, or Elk1 with both small- Administrative, technical, or material support (i.e., reporting or orga- molecule inhibitors and siRNA- or shRNA-mediated gene nizing data, constructing databases): A. Nagalingam, D. Sharma, N.K. Saxena Study supervision: A. Nagalingam, D. Sharma, N.K. Saxena silencing abolishes WFA-mediated DR5 induction. In conclusion, we uncovered a novel mechanism by which in vitro in vivo Grant Support WFA inhibits growth of breast cancer cells and , This work was supported by NIDDK NIH, K01DK076742, and which involves activation of RSK and ERK. We also demon- R03DK089130 (N.K. Saxena); NCI NIH R01CA131294, Avon Foundation, Breast Cancer Research Foundation (BCRF) 90047965 (D. Sharma), and NCI NIH strate a feed-forward loop of ERK and RSK that results in RO1 CA142604 (S.V. Singh). concurrent activation of CHOP and pElk1, their recruitment to The costs of publication of this article were defrayed in part by the payment DR5 promoter leading to DR5 activation. Our results, thus, of page charges. This article must therefore be hereby marked advertisement demonstrate the integral role of a previously unrecognized in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. functional cross-talk between WFA and ERK/RSK and CHOP/ Received July 22, 2013; revised January 16, 2014; accepted February 11, 2014; Elk1 axes in breast tumor growth inhibition. Also, our findings published OnlineFirst April 14, 2014.

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Mechanistic Elucidation of the Antitumor Properties of Withaferin A in Breast Cancer

Arumugam Nagalingam, Panjamurthy Kuppusamy, Shivendra V. Singh, et al.

Cancer Res Published OnlineFirst April 14, 2014.

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