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. Author manuscript; available in PMC: 2015 May 1.
Published in final edited form as: Cancer Res. 2014 Apr 14;74(9):2617–2629. doi: 10.1158/0008-5472.CAN-13-2081

Mechanistic elucidation of the antitumor properties of withaferin A in breast cancer

Arumugam Nagalingam 1, Panjamurthy Kuppusamy 2, Shivendra V Singh 3, Dipali Sharma 1,*, Neeraj K Saxena 2,*
PMCID: PMC4009451  NIHMSID: NIHMS571530  PMID: 24732433

Abstract

Withaferin A (WFA) is a steroidal lactone with antitumor effects manifested at multiple levels which are mechanistically obscure. Using a phospho-kinase screening array, we discovered that WFA activated phosphorylation of the S6 kinase RSK in breast cancer cells. Pursuing this observation, we defined activation of ERK-RSK and Elk1-CHOP kinase pathways in upregulating transcription of the death receptor DR5. Through this route, WFA acted as an effective DR5 activator capable of potentiating the biological effects of celecoxib, etoposide and TRAIL. Accordingly, WFA treatment inhibited breast tumor formation in xenograft and 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.

Keywords: Breast cancer, Elk1, RSK, Withaferin A, bioactive compound

Introduction

Breast cancer is the most commonly diagnosed cancer in women worldwide. Despite improvements in early diagnosis, and development of various targeted therapeutic approaches, breast cancer related mortality still remains at a high level. Major limitations associated with available therapeutic approaches are high toxicity, lower efficacy, therapeutic resistance and therapy-related morbidity. A more promising approach seems to be the development of more-effective, non-endocrine, non-toxic therapeutic strategies using active constitutive agents in natural products owing to their cancer preventive as well as therapeutic potential. Historically, natural products have played an important role in the discovery and development of novel anticancer agents (1, 2). Withania somnifera has been successfully used in traditional ayurvedic medicine to treat various diseases owing to its broad spectrum pharmacological efficacy (3–5). The root extract of Withania somnifera, is composed of 14 bioactive compounds known as Withanolides (6, 7). Withaferin A (WFA) is the most abundant and therapeutically effective withanolide and multiple studies have shown the anticancer activities of WFA (8–10). WFA administration decreases mammary tumors and pulmonary metastasis in MMTV-neu transgenic model and is associated with increased apoptosis (11) WFA-induced apoptosis involves reactive oxygen species (ROS) production (12), FOXO3a and Bim induction (8). While WFA effectively inhibits oncogenic transcription factors such as Stat3 (13), resulting in growth inhibition, it is interesting to note that WFA has also been reported to activate Notch signaling which plays an oncogenic role and is often hyperactive in breast cancer cells (14). Multiple other targets have been documented for WFA action such as NF-kB, BCL2, Hsp90, vimentin, and annexin II in various other cellular systems (15–20), 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 in breast cancer is needed to establish surrogate biomarkers for its efficacy and help in clinical development of this bioactive molecule, an issue we addressed by systematically elucidating the underlying mechanisms.

Because many cellular signaling events involve induced-phosphorylation of key targets, in the present study we utilized phosphokinase arrays to gain insight into the intricacies of WFA-mediated signaling networks and discovered that WFA induces RSK phosphorylation in breast cancer cells. We designed this study to examine the role of RSK and the underlying molecular mechanisms how WFA mediated activation of RSK leads to growth inhibition of breast cancer cells. Here, we provide strong evidence for ERK/RSK as an important mediator in WFA-induced breast cancer growth inhibition, and uncover a novel mechanism of WFA action through activation of ETS-like transcription factor 1 (Elk1)/C-EBP homology protein (CHOP) axis leading to DR5 activation.

Materials and Methods

Cell culture and reagents

The human breast cancer cell lines, MCF7, MDA-MB-231, T47D and MDA-MB-468 were obtained from the American Type Culture Collection (ATCC), resuscitated from early passage liquid nitrogen vapor stocks as needed and cultured according to supplier’s instructions. Cells were cultured for less than 3 months before reinitiating cultures and were routinely inspected microscopically for stable phenotype. Withaferin A (WFA) was procured from Calbiochem. Fluoromethyl ketone-methoxyethylamine (FMK-MEA), a specific p90RSK inhibitor was kindly provided by Dr. Jack Taunton (UCSF, Cellular and Molecular Pharmacology, San Francisco, CA) (21). U0126 and Trichostatin A (TSA) were procured from Sigma. CHOP overexpression construct was purchased from OriGene Technologies, Inc. Wild type and S383A mutant Elk1 plasmids were kindly provided by Dr. Andrew D. Sharrocks (University of Manchester, Manchester, UK) (22). ERK plasmids were kindly provided by Dr. Paul Shapiro, University of Maryland. Celecoxib was obtained from LKT laboratories. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was obtained from Millipore. Etoposide was purchased from Sigma.

Clonogenicity, anchorage-independent growth and cell-viability assays

To perform clonogenicity assay (23), breast cancer cells were treated with WFA as indicated for 10-days; colonies were counted. Anchorage-independent growth of breast cancer cells in the presence of WFA was assayed by colony formation in soft agar (24). Cell viability assay was performed using a commercially available XTT assay kit (Roche Applied Science, Indianapolis, IN).

Breast tumorigenesis assay

MDA-MB-231, MDA-MB-231-pLKO.1, MDA-MB-231-DR5shRNA1 and MDA-MB-231-DR5shRNA2 xenografts were generated as previously described (24), grouped in 2 experimental groups (8 mice/group) and treated with intraperitoneal injections of either vehicle (10% DMSO, 40% cremophor-EL, and 50% PBS) or vehicle containing 4 mg Withaferin A (ChromaDex Inc., Irvine, CA)/kg body weight 5days/week for 5 weeks. The dose and route of WFA administration were selected from previous study documenting in vivo efficacy of WFA (8). Tumors were collected after 4 weeks of treatment; measured, weighed, and subjected to further analysis by immunohistochemistry, RT-PCR and western blotting. At least four random, nonoverlapping representative images from each tumor section from eight tumors of each group were captured using ImagePro software for quantitation of pERK, pRSK, CHOP, pElk1, and DR5 expression. MMTV-neu mice model- Mammary tumor tissues from our previously published prevention study in MMTV-neu mice (11) were also used to determine the expression of these proteins by western blotting. In this study, WFA administration resulted in a statistically significant decrease in macroscopic mammary tumor size, microscopic mammary tumor area (11). All animal studies were in accordance with the guidelines of Johns Hopkins University IACUC and University of Pittsburgh IACUC.

Phospho-Antibody Array Analysis

Breast cancer cells were treated with WFA and the phospho-antibody array analysis was performed using the Proteome Profiler Human Phospho-Kinase Array Kit ARY003 from R&D Systems according to the manufacturer’s instructions. Array images were analyzed using the GeneTools image analysis software (Syngene).

Subcellular fractions, Immunoblotting, transfection, RNA interference, Immunofluorescence and confocal imaging

Cellular cytosolic and nuclear fractions were prepared following previously published protocol (25). Immunoblotting was carried out as described (26). The blots are representative of multiple independent experiments and bar diagrams are included showing quantitation of western blot signals. Breast cancer cells were transfected with ERK, CHOP, Elk1-WT and Elk1-S383A-mutant vectors using Lipofectamine-2000 (Invitrogen) and treated with WFA as indicated. For RNA interference, cells were transfected at 50% confluency with 100 nM of control siRNA or ERK1/2 siRNA (SignalSilence) using Oligofectamine (Cell Signaling Technology). Breast cancer cells were subjected to immunofluorescence analysis as described (24).

Chromatin immunoprecipitation (ChIP) and RNA isolation, RT-PCR

ChIP analyses were performed using our published procedure (27). RNA isolation, RT-PCR Total cellular RNA was extracted using the TRIZOL Reagent kit (Life Technologies, Inc., Rockville, MD). RT-PCR was performed using specific sense and antisense PCR primers.

Stable knockdown using Lentiviral short hairpin RNA

Five-six pre-made lentiviral DR5, CHOP and RSK short hairpin RNA (shRNA) constructs and a negative control construct created in the same vector system (pLKO.1) were purchased from Open Biosystems (Huntville, AL). Constructs were used for transient transfections using Fugene or Lipofectamine. Paired stable knockdown cells were generated following our previously established protocol (25).

Statistical Analysis

All experiments were performed thrice in triplicates. Statistical analysis was performed using Microsoft Excel software. Significant differences were analyzed using student’s t-test and two-tailed distribution. Results were considered to be statistically significant if p<0.05. Results were expressed as mean ± SE between triplicate experiments performed thrice.

Results

Withaferin A treatment inhibits clonogenicity, anchorage-independent growth of breast cancer cells and inhibits breast tumor progression in athymic nude mice

We first examined the effect of WFA on clonogenic potential and anchorage-independent growth of breast cancer cells. Treatment with 5 μM WFA resulted in ~50–60% inhibition in clonogenicity and soft-agar colony-formation, whereas higher concentrations were more inhibitory (Figure 1A, B). Exposure of breast cancer cells to WFA led to decreased cell viability (supplementary Figure 1A–D). WFA-mediated inhibition of cancer cell growth is associated with induction of apoptosis (8, 12, 17). Members of inhibitor-of-apoptosis protein (IAP) family, survivin and X-linked inhibitor-of-apoptosis protein (XIAP), mainly function to suppress apoptosis by inhibiting caspases (28) and are associated with increased aggressiveness, higher recurrence rate, and unfavorable breast cancer outcome (28–30). Decreased expression of survivin and XIAP was observed in breast cancer cells treated with WFA (Supplementary Figure 2A). An induction of cleaved-PARP was observed in the presence of 5 and 10 μM WFA (Supplementary Figure 2A). Treatment of breast cancer cells with 5 μM WFA for various time intervals showed a decrease in XIAP expression along with an induction of PARP cleavage (Supplementary Figure 2B).

Figure 1. WFA inhibits clonogenicity, anchorage-independent growth and breast tumor growth in nude mice.

Figure 1

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(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 three weeks. Histogram represents average number of colonies counted (in six micro-fields). *, P<0.001, compared with untreated controls. Vehicle-treated cells, denoted with the letter “C”. (C) MDA-MB-231 cells 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 = 8 mice per group); (P< 0.001). (D, E) Tumors from vehicle (V) and WFA-treated mice were subjected to immunohistochemical (IHC) analysis using Ki67, Survivin and XIAP antibodies. Bar diagrams show quantitation of IHC-analysis. Columns, mean (n =8); bar, SD. * significantly different (P< 0.005) compared with control. (F) TUNEL-positive cells in tumor sections were counted. Each bar represents the mean (n=6–8). *, P<0.01, compared with untreated controls.

We further investigated the in vivo physiological relevance of our in vitro findings by evaluating whether WFA had inhibitory effects on the development of breast carcinoma in nude mouse models. Tumor growth was significantly inhibited in WFA-treated experimental group in comparison to the control group (Figure 1C). Ki-67, a nuclear non-histone protein, is one of the major markers of tumor proliferation (31) used as a decision-making tool for adjuvant therapy (32). The immunohistochemical assessment of tumor proliferation showed higher Ki-67 in the control group as compared with the WFA-treated group (Figure 1D, E). In our in vitro analysis, we found that WFA modulated the expression of survivin and XIAP in breast cancer cells. Corroborating the in vitro findings, the tumors from the WFA-treated mice exhibited lower expression of survivin and XIAP (Figure 1D, E). The number of TUNEL-positive apoptotic cells was increased in tumors from the WFA-treated mice compared with vehicle control group (Figure 1F). Collectively, these results show that WFA treatment results in suppression of tumor growth, inhibition of cellular proliferation and increased apoptosis in the breast tumors.

Withaferin A dependent changes in phosphorylation of signaling mediators in breast cancer cells

Phosphorylation of kinases is fundamentally important to multiple aspects of signaling-networks and cellular functions. To identify cellular signal-transduction pathways involved in WFA-induced inhibition of breast carcinogenesis, we interrogated 46 specific Ser/Thr/Tyr phosphorylation sites of 38 selected proteins using phosphoprotein arrays. Breast cancer cells were treated with 5 μM WFA for 3h and subjected to phospho-protein analysis. Phosphorylation level of p90-ribosomal S6 kinase (RSK) was significantly increased in both MCF7 and MDA-MB-231 cells. In addition, WFA treatment increased phosphorylation of ERK in breast cancer cells (Figure 2A, B and Supplementary Figure 3). Dysregulated RSK expression or activity has been associated with several human cancers, controlling various downstream signaling pathways and modulating cellular processes (33, 34). In contrast to the conventional oncogenic role of RSK, multiple studies have shown RSK as a potential tumor suppressor, a participant in p53-dependent cell growth arrest, showing an important role in decreasing cancer cell proliferation, invasion and metastasis (33–35). We further explored the molecular mechanisms how WFA activates RSK and the biological significance of RSK-activation in WFA-mediated inhibition of breast carcinogenesis.

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.

Figure 2

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(A, B) MCF7 and MDA-MB-231 breast cancer cells were treated with 5 μM 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 phosphorylated-RSK-Ser380 (pRSK) and total RSK in breast cancer cells treated with WFA as indicated. (D) Breast cancer cells were treated with 5 μM 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 phosphorylated-ERK44/42 (pERK) 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 48h followed by immunoblot analysis of ERK levels. (H) Breast cancer cells were transfected with siERK as in G, followed by WFA treatment (5 μM, 3h) and immunoblot analysis of pRSK and total RSK levels. Vehicle-treated cells are denoted with C.

Withaferin A treatment activates p90-ribosomal S6 kinase (RSK) via extracellular signal-regulated kinase (ERK)-dependent manner in breast cancer cells

To confirm whether WFA can activate RSK as indicated by phosphoprotein array results, and to clarify its dose- and time-dependent response, we treated breast cancer cells with WFA. Immunoblot analysis showed that phosphorylation of RSK increased in a concentration-dependent manner in both MCF7 and MDA-MB-231 cells (Figure 2C). Treatment of breast cancer cells with 5 μM WFA for various time-intervals exhibited striking increase in RSK phosphorylation within 30 minutes (Figure 2D, Supplementary Figure 4A) whereas total RSK remained unchanged. RSK activation is controlled by canonical Ras/mitogen-activated protein kinase (MAPK) pathway via direct phosphorylation of RSK by ERK (34). ERK itself represents a major survival signaling pathway that promotes cancer cell survival via inhibiting apoptosis, however, a proapoptotic role of ERK signaling has also been shown (36). Our phosphoprotein array analysis showed elevated phosphorylation of ERK in breast cancer cells treated with WFA. Because of these interesting attributes, we explored the effect of WFA on ERK activation. Increased phosphorylation of ERK was observed in response to WFA treatment as compared to untreated cells (Figure 2E). Breast cancer cells, treated with 5 μM WFA for various intervals of time, showed that WFA increased ERK phosphorylation within 30 minutes of WFA treatment (Figure 2F, Supplementary Figure 4B). We questioned whether ERK is a key player in WFA-mediated RSK activation and towards this end, we utilized ERK siRNA to silence ERK (Figure 2G) and then investigated its impact on WFA-induced RSK activation. Immunoblot analysis for phosphorylated RSK levels after ERK silencing and WFA treatment showed that indeed ERK silencing inhibited WFA-induced RSK phosphorylation (Figure 2H, Supplementary Figure 4C). Inhibition of ERK phosphorylation using U0126 also resulted in abrogation of WFA-mediated RSK activation (Supplementary Figure 5A). Next, we examined the importance of ERK and RSK activation in WFA-mediated apoptosis in breast cancer cells utilizing MEK/ERK inhibitor, U0126 and RSK inhibitor, fmk-MEA. WFA treatment resulted in elevated PARP-cleavage indicating increased apoptotic response in breast cancer cells which was inhibited in breast cancer cells co-treated with U0126 or fmk-MEA (Supplementary Figure 5B, C). ERK silencing abrogated WFA-mediated inhibition of clonogenicity of breast cancer cells and reintroduction of ERK in ERK-silenced cells resensitized them to WFA (Supplementary Figure 6A). RSK inhibition using RSK-shRNA (s) (Supplementary Figure 6B) also rendered breast cancer cells non-responsive to WFA-mediated inhibition of clonogenicity of breast cancer cells (Supplementary Figure 6C). Collectively, these evidences support the notion that RSK and ERK activation play an important role in mediating biological effects of WFA in breast cancer cells.

Withaferin A induces concomitant upregulation and nuclear translocation of Elk1 and CHOP via ERK/RSK signaling axis in breast cancer cells

The ETS domain transactivation factor Elk1 is a direct target of the MAP kinase pathways and phosphorylation of the Elk1 transcriptional-activation domain by MAP kinases triggers its activation which further induces apoptosis and growth inhibition (37). Phosphorylation at serine 383 plays a critical role in Elk1 activation. Increased Elk1 phosphorylation was observed within 30 minutes of WFA treatment which was diminished after 6h post-treatment (Figure 3A, B). We examined whether WFA-induced Elk1 phosphorylation involves ERK. Indeed, pretreatment of breast cancer cells with ERK siRNA reduced WFA-induced phosphorylation of Elk1 indicating a direct regulatory role of ERK (Supplementary Figure 7A, B). Our studies discovered the involvement of RSK in WFA function therefore we investigated whether RSK is integral for WFA-mediated Elk1 phosphorylation. RSK-specific inhibitor fmk-MEA inhibited phosphorylation of RSK (Figure 3C). Pretreatment of breast cancer cells with fmk-MEA followed by WFA treatment showed that inhibition of RSK phosphorylation inhibited WFA-induced Elk1 phosphorylation (Figure 3D, Supplementary Figure 7C). Phosphorylation can also affect the sub-cellular localization of Ets proteins (37, 38). As evident in Figure 3E, WFA treatment did not affect the total expression level of Elk1 in either cytoplasmic or nuclear fraction, however, increased level of phosphorylated Elk1 was observed in nuclear fraction within 30 minutes post-treatment which remained elevated for the duration of the treatment (Figure 3E, Supplementary Figure 7D). Immunofluorescence analysis of breast cancer cells showed nuclear phosphorylated Elk1 in WFA-treated cells (Figure 3F).

Figure 3. RSK signaling axis mediates WFA-induced phosphorylation of Elk1.

Figure 3

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(A) Immunoblot analysis of phosphorylated-Elk1 (pElk1) and Elk1 in breast cancer cells treated with 5 μM WFA for indicated time intervals. (B) Bar diagram shows 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 μM) for indicated time-intervals. Total lysates were immunoblotted for pRSK and total RSK expression. (D) Breast cancer cells were pre-treated with fmk-MEA (6 μM, 3h) followed by WFA treatment (5 μM, 3h) 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 μM WFA for indicated time-intervals. (F) Breast cancer cells were treated with 5 μM WFA for 6h and 24h as indicated and subjected to immunofluorescence analysis of pElk1. Nuclei were visualized with DAPI staining. Vehicle-treated cells are denoted with ‘C’.

Induction of apoptotic response by many pharmacologically active compounds and anticancer drugs is achieved by activation of endoplasmic reticulum (ER) stress. ER stress signaling system induces transcription of C/EBP homologous protein (CHOP), a key proapoptotic transcription factor that binds with other transcription factors and induces pro-apoptotic genes (39). We found that WFA increased expression of CHOP in a temporal manner with a significant increase observed within 30 minutes post-treatment (Figure 4A, B). To evaluate the contribution of ERK in WFA-induced CHOP overexpression, we examined CHOP expression after ERK silencing using ERK siRNA. Results indicated that indeed ERK silencing abrogated WFA-induced CHOP expression (Figure 4C, D) suggesting the involvement of ERK in the CHOP-stimulatory effect of WFA. Once upregulated, CHOP translocates to the nucleus and participates in transcriptional modulation of responsive genes critical for tumor cell apoptosis (39). Analysis of nuclear and cytoplasmic fractions from WFA-treated cells showed that WFA treatment increased nuclear accumulation of CHOP (Figure 4E, F). Immunostaining of MCF7 and MDA-MB-231 cells showed that WFA treatment increases nuclear accumulation of CHOP whereas untreated cells showed predominantly cytoplasmic localization of CHOP (Figure 4G). CHOP inhibition using CHOP-shRNA (s) (Supplementary Figure 8A) rendered breast cancer cells non-responsive to WFA-mediated inhibition of clonogenicity of breast cancer cells (Supplementary Figure 8B) exhibiting importance of CHOP in WFA function. These findings suggest that ERK/RSK signaling axis plays an important role in regulating upregulation and activation of CHOP and Elk1 in breast cancer cells.

Figure 4. WFA increases CHOP expression via ERK and promotes its nuclear translocation.

Figure 4

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(A) Immunoblot analysis of CHOP in breast cancer cells treated with 5 μM WFA for indicated time-intervals. (B) Bar diagram shows 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 48h followed by WFA (5 μM, 3h) treatment and immunoblot analysis using anti-CHOP antibodies. Control transfected cells are denoted with C. (D) Bar diagram shows 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 μM WFA for indicated time-intervals. (F) Bar diagram shows quantitation of western blot signals from multiple independent experiments. *, P<0.05, compared with untreated controls. (G) Breast cancer cells were treated with 5 μM WFA for 6h and 24h and subjected to immunofluorescence analysis of CHOP. Nuclei were visualized with DAPI staining. Vehicle-treated cells are denoted with “C”.

Elk1 and CHOP contribute to withaferin A-induced upregulation of Death receptor 5 in breast cancer cells

Death receptor 5 (DR5) is a death domain-containing transmembrane receptor that triggers apoptotic response upon overexpression or activation by certain stimuli, including small molecule anticancer drugs and bioactive molecules (40, 41). We found that WFA induced the expression of DR5 in breast cancer cells (Figure 5A, Supplementary Figure 9 A–D). The fact that DR5 promoter contains cis-acting CHOP-like binding sequence prompted us to explore the connection between WFA-mediated CHOP-induction and DR5-upregulation. We investigated whether enforced overexpression of CHOP (Figure 5B) using CHOP overexpression construct alters WFA-mediated DR5 expression. Overexpression of CHOP in MCF7 and MDA-MB-231 cells increased DR5 expression potentiating the effect of WFA (Figure 5C, Supplementary Figure 9E). In a reciprocal approach, CHOP was silenced using CHOP-shRNA approach (Supplementary Figure 8A) followed by WFA treatment. Silencing of CHOP in breast cancer cells inhibited WFA-mediated induction of DR5 expression (Figure 5D). A recent study reported that DR5 promoter contains a putative Elk1 binding site and mutations in this binding site affected DR5 transactivation suggesting a link between DR5 and Elk1 (42). Because there was no previous study linking Elk1 to DR5 expression in breast cancer cells, we asked whether Elk1 was indeed involved in regulating WFA-mediated DR5 expression. Transactivation potential of Elk1 is enhanced by S383 phosphorylation (38) which is increased by WFA (Figure 3A, B), therefore, we focused on the key phosphoacceptor-residue Ser383. Breast cancer cells were transfected with wild-type or phospho-deficient (S383A) Elk1 constructs. Expression of wild-type Elk1 increased DR5 expression whereas phospho-deficient Elk1 did not affect DR5 expression (Figure 5E). To examine whether Elk1 contributes to WFA-mediated DR5 upregulation, we transfected breast cancer cells with wild-type or phospho-deficient Elk1 constructs followed by WFA treatment. Wild-type Elk1 potentiated whereas phospho-deficient Elk1 inhibited WFA-mediated DR5 expression (Figure 5F, Supplementary Figure 9F) demonstrating the important role of p-Ser383-Elk1 in WFA action. Our studies also showed an important role of RSK phosphorylation in WFA-mediated Elk1 phosphorylation (Figure 3D, Supplementary Figure 7C) therefore we examined whether RSK inhibitor fmk-MEA could modulate WFA-mediated DR5 upregulation. Indeed, inhibition of RSK phosphorylation resulted in inhibition of WFA-induced DR5 expression (Figure 5G). RSK was silenced using RSK-shRNA approach (Supplementary Figure 6B) followed by WFA treatment. Silencing of RSK in breast cancer cells inhibited WFA-mediated induction of DR5 expression (Figure 5H). In an effort to better understand the molecular events involved in WFA-induced DR5 expression, we used chromatin immunoprecipitation (ChIP) analyses to examine the recruitment of pElk1 and CHOP to the DR5 promoter. pElk1 and CHOP were associated with the DR5 promoter in the presence of WFA. DR5 in repressed state has been shown to be associated with HDAC1 (43). Intriguingly, we observed release of HDAC1 from DR5 promoter in response to WFA treatment and a significant increase in histone H4 acetylation indicating active chromatin conformation (Figure 5I, Supplementary Figure 10A). Treatment of breast cancer cells with HDAC inhibitor, TSA, resulted in increased expression of DR5 (Supplementary Figure 10B) supporting a functional role of HDAC1 in regulation of DR5 expression. Taken together, these data show that WFA induces DR5 expression in breast cancer cells through a mechanism involving CHOP and Elk1.

Figure 5. CHOP and Elk1 contribute to WFA-induced upregulation of Death receptor 5 in breast cancer cells.

Figure 5

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(A) Immunoblot analysis of DR5 in breast cancer cells treated with 5 μM 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 μM, 3h) treatment (CHOPO/E+WFA). (D) Breast cancer cells were transfected with CHOP-shRNA1 followed by WFA (5 μM, 3h) 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 phosphorylated Elk1 (pElk1), total Elk1 and DR5 expression. (F) MCF7 cells were transfected with Elk1 WT and Elk1M as in E, followed by WFA (5 μM, 3h) treatment. Untransfected controls are denoted with C. Total lysates were immunoblotted for pElk1, total Elk1 and DR5 expression. (G) Immunoblot analysis of phosphorylated-RSK, total RSK and DR5 in MDA-MB-231 cells pre-treated with fmk-MEA (6 μM, 3h) followed by WFA (5 μM, 3h) treatment. (H) Breast cancer cells were transfected with RSK-shRNA1 followed by WFA (5 μM, 3h) treatment. Total lysates were analyzed for DR5 expression. (I) MCF7 cells were treated with 5 μM WFA for 3h and 6h and subjected to chromatin immunoprecipitation 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. Vehicle-treated cells are denoted with ‘C’.

Withaferin A is a potent inducer of Death receptor 5 and silencing of Death Receptor 5 abrogates withaferin A-mediated growth-inhibition of breast cancer cells

DR5, upon induction, mediates enhancement of TRAIL-induced apoptosis and contributes to apoptosis by other small molecule drugs (44). Using established small-molecule DR5-inducers (TRAIL, celecoxib and etoposide), we compared the efficacy of WFA to induce DR5 expression in breast cancer cells. It was interesting to note that WFA treatment resulted in a greater induction of DR5 expression in comparison to TRAIL, celecoxib and etoposide (Figure 6A, Supplementary Figure 11A). To determine whether WFA enhances the effect of small-molecule-DR5 inducers, we treated breast cancer cells with WFA in combination with TRAIL, etoposide and celecoxib and assessed modulation of DR5 expression, clonogenicity and soft-agar colony formation. Combination treatment with WFA enhanced etoposide, celecoxib and TRAIL-induced DR5 expression (Figure 6B, Supplementary Figure 11B) and resulted in significantly higher inhibition of clonogenicity and soft-agar colony formation (Figure 6C, D). These results show that WFA is a potent inducer of DR5 which can act as an effective bioactive alternative to TRAIL, celecoxib and etoposide as well as enhance their effect when used in combination.

Figure 6. WFA is a potent inducer of Death receptor 5 in breast cancer cells and stable knockdown of Death Receptor 5 abrogates withaferin A-mediated inhibition of growth of breast cancer cells.

Figure 6

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(A) Immunoblot analysis of DR5 in MCF7 cells treated with 50 μM Celecoxib (CCB), 1 μM Etoposide (E), 100 ng/ml TRAIL (T) and 5 μM withaferin A (WFA) for 6h. (B) MCF7 cells were treated with CCB, Etoposide, TRAIL alone as in A or in combination with 5 μM withaferin A (WFA) for 6h. 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 three weeks. Results are expressed as average number of colonies counted (in six micro-fields). *, P<0.001, compared with untreated controls; #, P<0.001, compared with E treated cells; **, P<0.005, compared with CCB treated cells; ***, P<0.001, compared with T treated cells. Vehicle-treated cells are denoted with the letter “C”. (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 5μM 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 (5μM). (H, 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 (5μM). Results are expressed as average number of colonies counted (in six micro-fields). *, P< 0.001, compared with untreated pLKO.1 cells; #, P< 0.005, compared with untreated DR5shRNA cells. (J, K, L) MDA-MB-231-pLKO.1, MDA-MB-231-DR5shRNA1 and MDA-MB-231-DR5shRNA2 cells 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 = 8 mice per group); (P< 0.001). Representative tumor images are shown here. Tumor weight is shown in the table.

To directly examine the role of DR5 in WFA-mediated growth inhibition of breast cancer cells, we used DR5shRNA lentiviruses and puromycin to select for stable pools of MCF7 and MDA-MB-231 cells with DR5 depletion. We analyzed pLKO.1 and DR5shRNA stable MCF7 and MDA-MB-231 cell pools for DR5 protein expression, and found that DR5 protein expression was significantly knocked-down in DR5shRNA 1–3 cells as compared to pLKO.1 control cells (Figure 6E). WFA increased PARP cleavage in pLKO.1 cells whereas no change in cleaved PARP was observed in DR5shRNA MCF7 and DR5shRNA MDA-MB-231 cells (Figure 6F). WFA treatment efficiently inhibited clonogenicity and soft-agar-colony formation of pLKO.1 breast cancer cells but not of DR5shRNA cells (Figure 6G–I). We further investigated the in vivo physiological relevance of our in vitro findings by evaluating whether DR5 is integral for the inhibitory effects of WFA on the development of breast carcinoma in nude mouse models. MDA-MB-231-pLKO.1 and MDA-MB-231-DR5shRNA1 and shRNA2 were utilized in xenograft-athymic nude mice model. Tumor growth was significantly inhibited in WFA-treated MDA-MB-231-pLKO.1 (vector control group) whereas WFA was unable to inhibit tumor growth in MDA-MB-231-DR5shRNA1 and shRNA2 groups (Figure 6J, K, and L). These results showed that WFA-induced DR5 overexpression is indeed a crucial component of the signaling machinery used by WFA in inhibiting growth of breast cancer cells.

Withaferin A administration modulates activation of ERK/RSK and CHOP/Elk1 axes in vivo

Our studies show that WFA treatment inhibits breast tumor progression in vivo (Figure 1C). We utilized tumor tissue from the same experiment to determine the effect of WFA treatment on the expression and activation of important signaling molecules. Tumors from WFA-treated mice exhibited increased phosphorylation of ERK, RSK, Elk1 as well as higher expression CHOP and DR5 in comparison to the vehicle-treated group (Figure 7A). Also, WFA treated tumors showed transcriptional upregulation of DR5 (Figure 7B). Immunohistochemical analysis showed that tumors from WFA-treated mice exhibited higher number of tumor cells showing increased expression of pERK, pRSK, CHOP, pELK1 and DR5 as compared to tumors from vehicle-treated group (Figure 7C, D) providing physiological relevance to our in vitro findings. Earlier studies from our group have shown that WFA treatment prevents mammary cancer development in MMTV-neu mouse model, tumor weight in the WFA-treatment group was lower by 50% in comparison with the control group (11). Mammary tumor tissues from this study were utilized to examine ERK/RSK signaling axis and DR5. Tumors from WFA-treated group exhibited increased expression of phosphorylated ERK and RSK as well as higher expression of DR5 in comparison to control group (Figure 7E). Collectively, the findings presented here suggest that WFA inhibits breast tumor progression and provide in vitro as well as in vivo evidence for the involvement of RSK as an important mediator, and uncover a novel mechanism of WFA action through activation of Elk1 leading to DR5 activation.

Figure 7. In vivo evidence for WFA-mediated activation of ERK/RSK and CHOP/Elk1 axes.

Figure 7

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(A) Western blot analysis of indicated protein levels in MDA-MB-231 cells-derived tumors developed in nude mice and treated with vehicle or WFA for five weeks. (B) Total RNA was isolated from tumor samples and expression of DR5 was analyzed using RT-PCR analysis. (C, D) Tumors were subjected to immunohistochemical analysis using pERK, pRSK, CHOP, pELK1 and DR5 antibodies. Bar diagrams show quantitation of protein expression in tumors from vehicle and WFA-treated mice. Columns, mean (n =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.

Discussion

The realization of the ability of bioactive small-molecule agent withaferin A (WFA) to effectively inhibit carcinogenesis in a non-toxic, non-endocrine manner has made this agent of potential interest in the treatment of breast cancer and sparked a new interest in understanding the underlying molecular mechanisms. Deciphering the key nodes of WFA action can help in establishing surrogate biomarkers for its efficacy and help in clinical development of this bioactive molecule. We found that WFA effectively inhibits the growth of breast cancer cells in vitro and in vivo but our phosphokinase array studies led us to novel discoveries that extend beyond the popular proliferative and oncogenic role of certain kinases and inhibition of these kinases by anti-cancer agents to achieve tumor growth inhibition. Both p90-ribosomal S6 kinase (RSK) and ERK are popularly known for their oncogenic role but this study illustrates that WFA despite efficiently inhibiting breast tumor growth, increases phosphorylation of RSK in breast cancer cells via activation of ERK. These studies provide an interesting mechanism by which WFA-induced ERK/RSK, in a concerted action, results in concomitant upregulation/activation of CHOP and Elk1 (ETS-like transcription factor 1). We also identify WFA as a potent DR5 activator in breast cancer whose effects mimic molecular and cell-biological outcomes of ligand-dependent DR5 activation. Mechanistically, this process is associated with recruitment of CHOP and Elk1 to DR5 promoter, increased histone acetylation in conjunction with clearance of histone deacetylase HDAC1. In vivo analyses of tumor xenografts and tumors from MMTV-neu mice provide further evidence of involvement of ERK/RSK and CHOP/Elk1 axes and an integral role of DR5. Based on these data, we provide a schematic diagram depicting a series of events including a feed-forward interaction of ERK and RSK, direct involvement of CHOP/Elk1/HDAC1 in DR5 upregulation, which is operative in WFA-induced DR5-dependent growth-inhibition of breast cancer cells (Figure 7F).

Our studies offer the first evidence of the ability of WFA to activate RSK. RSK directly phosphorylates transcription factors hence regulating the transactivation of multiple gene targets. RSK positively regulates diverse cellular processes, including transformation, cell cycle, cell proliferation, cell survival, and migration in response to various growth factors, chemokines and other stimuli (45). RSK has been implicated in apoptosis inhibition achieved in part by phosphorylation of Bad, CEBPβ and DAPK (34), as well as apoptosis induction via phosphorylating Nur77 (46). We show that the ability of RSK to mediate activation of Elk1 and CHOP leading to upregulation of DR5 is critical to WFA outcomes in breast cancer cells. This provides a new mechanism by which WFA can achieve effective breast tumor growth-inhibition. Also, these discoveries have potentially important implications in regards to increasing interest in targeting breast cancer through manipulation of DR5.

Activation of death receptors to induce apoptosis in tumor cells has been recognized as an effective way to therapeutically target epithelial-cell derived cancers but this notion is marred by the toxic effects of death receptor ligands, FASL and TNF. Over the few years, several small molecule activators of the death receptor family have been reported with some more potent than others (44). WFA has been shown to sensitize TRAIL-induced apoptosis and activates DR5 expression in human renal cancer cells (Caki cells, but not in human normal mesangial cells) (41). Here, we show that WFA is more effective than three other known DR5 activators, TRAIL, etoposide and celecoxib, in activation of DR5 in breast cancer cells hence, appears to be a good candidate for further development as an effective DR5 inducer. It is interesting to note that in recent years, many phase 1 and 2 single agent and combination clinical trials have been conducted to examine the efficacy and safety of recombinant human TRAIL (dulanermin, (Amgen/Genentech)), and the agonistic monoclonal antibodies to DR5 (lexatumumab (Human Genome Sciences), conatumumab (Amgen), drozitumab (Genentech), tigatuzumab (Daiichi-Sankyo) and LBY135 (Novartis)) (44, 47). Studies with these pro-apoptotic receptor agonists (PARAs) have been encouraging but no full phase 3 studies have been performed suggesting the need for novel PARAs with improved properties.

Moreover, we report a novel finding that ERK activation is important for WFA action in breast cancer cells. Generally considered a survival-signaling pathway, ERK-mediated induction of apoptotic pathways has also been reported (36, 48). Kinetics and duration of ERK activation are important for these unusual effects of ERK. A transient activation of ERK inhibits cell death (49) whereas prolonged ERK activation is associated with the proapoptotic effect of ERK (36). WFA induces a sustained ERK activation in breast cancer cells which was observed for the duration of experiment (till 24 hours). Very recently, it was reported that WFA increases ERK phosphorylation in MCF7 and SUM159 cells (50). It is known that the ERK protein directly phosphorylates Elk1 (38). Hence, the finding of the involvement of ERK in regulation of WFA-induced cell death resulted in our subsequent novel finding that WFA activates Elk1, which contributes to DR5 induction. In our study, inhibition of ERK, RSK or Elk1 with both small molecule inhibitors and siRNA –or shRNA-mediated gene silencing abolishes WFA-mediated DR5 induction.

In conclusion, we uncovered a novel mechanism by which withaferin A inhibits growth of breast cancer cells in vitro and in vivo, which involves activation of RSK and ERK. We also demonstrate a feed-forward loop of ERK and RSK that results in concurrent activation of CHOP and pElk1, their recruitment to DR5 promoter leading to DR5 activation. Our results thus demonstrate the integral role of a previously unrecognized functional crosstalk between withaferin A and ERK/RSK and CHOP/Elk1 axes in breast tumor growth inhibition. Also, our findings may potentially open new avenues of research on the role of withaferin A as a novel pro-apoptotic receptor agonist (PARA).

Supplementary Material

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Acknowledgments

Financial Support: This work was supported by NIDDK NIH, K01DK076742 and R03DK089130 (to NKS); NCI NIH R01CA131294, Avon Foundation, Breast Cancer Research Foundation (BCRF) 90047965 (to DS), and NCI NIH RO1 CA142604 (to SVS).

Footnotes

Conflict of Interest: N/A

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Supplementary Materials

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