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. 2013 Jul;15(7):738–748. doi: 10.1593/neo.121888

PAX3-FOXO1 Induces Up-Regulation of Noxa Sensitizing Alveolar Rhabdomyosarcoma Cells to Apoptosis1,2

Amy D Marshall *,†, Fabrizio Picchione *, Ramon I Klein Geltink *, Gerard C Grosveld *
PMCID: PMC3689237  PMID: 23814486

Abstract

Alveolar rhabdomyosarcoma (ARMS) has a much poorer prognosis than the more common embryonal subtype. Most ARMS tumors characteristically possess a specific genomic translocation between the genes of PAX3/7 and FOXO1 (FKHR), which forms fusion proteins possessing the DNA binding domains of PAX3/7 and the more transcriptionally potent transactivation domain of FOXO1. We have shown that the proapoptotic BH3-only family member Noxa is upregulated by the PAX3-FOXO1 fusion transcription factor in a p53-independent manner. The increased expression of Noxa renders PAX3-FOXO1-expressing cells more susceptible to apoptosis induced by a γ-secretase inhibitor (GSI1, Z-LLNle-CHO), the proteasome inhibitor bortezomib, and BH3 mimetic ABT-737. Apoptosis in response to bortezomib can be overcome by shRNA knockdown of Noxa. In vivo treatment with bortezomib reduced the growth of tumors derived from a PAX3-FOXO1-expressing primary myoblast tumor model and RH41 xenografts. We therefore demonstrate that PAX3-FOXO1 up-regulation of Noxa represents an unanticipated aspect of ARMS tumor biology that creates a therapeutic window to allow induction of apoptosis in ARMS cells.

Introduction

Rhabdomyosarcoma (RMS) is the most prevalent soft tissue sarcoma in children. RMS is thought to be derived from cells of mesenchymal lineage and tumors express muscle-specific markers such as MYO-D, desmin, myoglobin, and proteins of the contractile apparatus. RMS consists of two main histologic cell types, embryonal and alveolar RMS (ERMS and ARMS, respectively). ERMS is thought to resemble embryonic developing muscle, while ARMS contains alveolar structures resembling lung tissue in appearance [1]. Of these two subtypes, ARMS is the most aggressive and has the poorest prognosis [1,2].

ARMS tumors are associated with chromosomal translocations between the PAX3 or PAX7 and FOXO1 genes in approximately 55% and 22% of cases, respectively [3]. In addition, other similar rare trans-locations of the PAX3 gene to that of other transcription factors (AFX, NCOA1, and NCOA2) have been identified in ARMS [4–6]. Fusion of the PAX3 DNA binding domain to the more potent transactivation domain of FOXO1 results in a fused transcription factor with stronger transcriptional activity than wild-type PAX3 [7]. Within the ARMS subtype, tumors expressing the PAX3-FOXO1 fusion protein are associated with the poorest prognosis, particularly if the patient presents with metastatic disease [3]. Moreover, a single copy of PAX3-FOXO1 is usually found in ARMS, while PAX7-FOXO1 often shows genomic amplification of the fusion allele [8], supporting the notion that PAX3-FOXO1 is a more potent oncogene than PAX7-FOXO1. Therefore, we have identified genes regulated by PAX3-FOXO1 to determine if these could represent viable novel therapeutic targets for the treatment of ARMS [9,10].

In the treatment of more aggressive cancer types, which are resistant to traditional chemotherapeutics, new strategies are being developed to target these diseases. Several new drugs are showing promise in a variety of different cancer types at specifically inducing apoptosis in cancer cells. Bortezomib (PS-341, Velcade) is a potent 26 S proteasome inhibitor, which causes the accumulation of misfolded or unfolded proteins in the endoplasmic reticulum, inducing endoplasmic reticulum stress. The accumulation of these unfolded proteins results in the unfolded protein response [11]. Bortezomib treatment also stabilizes proapoptotic factors that are normally degraded through the proteasome [12]. The cumulative effect of bortezomib treatment is induction of apoptosis. Bortezomib is currently approved by the Food and Drug Administration (FDA) for treatment of multiple myeloma and refractory mantle cell lymphoma, and phase II trials are underway for multiple other cancer types including solid tumors [13]. Another potential cancer therapy consists of γ-secretase inhibitors (GSIs), which were originally designed for the treatment of Alzheimer's disease [14] but have since been investigated as potential cancer therapies to target tumor cells with high Notch expression [15]. Though they may show promise in some tumor types that are not Notch dependent [16], GSIs have been found to cause severe gastrointestinal toxicity due to goblet cell metaplasia induced by Notch inhibition [17]. Recent findings, however, have shown that co-treatment with glucocorticoids can protect the gut of mice from GSI toxicity [18], renewing interest in GSIs as anticancer therapeutics. Both GSI1 (Z-LLNle-CHO) and bortezomib have been shown to induce melanoma cell apoptosis while sparing normal melanocytes. Both compounds specifically induce the mRNA and protein expression of the proapoptotic BH3-only factor, Noxa/Pmaip1, which in turn induces mitochondrial-based apoptosis in a p53-independent manner [19,20]. The effect of GSI1 on RMS has not, to the author's knowledge, been investigated in ARMS. However, bortezomib has been shown to induce apoptosis in some ERMS- and ARMS-derived cell lines and reduce growth of some tumors in a xenograft model [21,22].

In this study, we have found that the ARMS-associated PAX3-FOXO1 fusion oncogene specifically upregulates proapoptotic BH3-only factor Noxa, in a p53-independent manner. Up-regulation of Noxa by PAX3-FOXO1 sensitizes the cells to treatment with GSI1 and bortezomib that also induces Noxa-dependent, p53-independent, apoptosis. In addition, apoptosis is induced in PAX3-FOXO1 cells treated with ABT-737 that targets antiapoptotic Bcl-2, Bcl-xL, and Bcl-w, while Noxa inhibits antiapoptotic MCL-1, representing a dual approach to induce mitochondrial apoptosis specifically in PAX3-FOXO1-expressing cells. Treatment with bortezomib was sufficient to reduce the growth of tumorigenic primary mouse myoblasts expressing PAX3-FOXO1 and human RH41 ARMS xenografts in vivo. Thus, we have illustrated the validity of such Noxa-inducing factors as potential therapies, specifically targeted to fusion-positive ARMS tumors.

Materials and Methods

Cell Culture

Primary myoblasts were isolated from 1- to 5-day-old C57Bl6/J, p53-/- [23] or wild-type control, or Arf-/- [24] mice and cultured according to Marshall et al. [9]. RMS lines were cultured in Dulbecco's modified Eagle's medium and 10% Cosmic calf serum following standard procedures. These cell lines were obtained from Dr Houghton (Nationwide Children's Hospital) in 2005 and were authenticated by Western blot to express PAX3-FOXO1 and skeletal muscle markers (data not shown). Human skeletal muscle myoblasts (HSMMs; Lonza, Allendale, NJ) were cultured according to the supplier's instructions. HSMMs were obtained from Lonza and have undergone less than 10 passages and were authenticated by Western blot to express skeletal muscle markers (data not shown).

Cells were transduced with retroviral constructs packaged using phoenix Eco cells [25] consisting of MSCV backbone with PAX3 or PAX3-FOXO1 followed by an internal ribosome entry site-green fluorescent protein (IRES-GFP) or an SV40 promoter-puromycin gene to allow selection of transduced cells by fluorescence-activated cell sorting (FACS) or puromycin treatment, respectively. shNoxa targeted the sequence 5′-CAACACTGAATGTTCTAGTGAA-3′ in the context of pre-miR-30 sequences to ensure correct processing.

Real-Time Polymerase Chain Reaction

RNA was isolated by TRIzol (Invitrogen), and reverse transcription-polymerase chain reaction (PCR) was performed using the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen, Green Island, NY) according to the manufacturer's protocol. Real-time PCR was then performed using TaqMan Universal PCR Master Mix (Applied Biosystems, Green Island, NY), 0.3 µM primers, and 0.2 µM probes as follows: mNoxa Fwd, 5′-CTGTGGTTCTGGCGCAGAT-3′; mNoxa Rev, 5′-TGGCTGTATCTCTCCACAAGTTCT-3′; mNoxa Probe, 5′-CTGGGAAGTCGCAAAA-3′ (5′-FAM/3′-BHQ1); hNoxa Fwd, 5′-GAGCTGGAAGTCGAGTGTGCTA-3′; hNoxa Rev, 5′-TGCC-GGAAGTTCAGTTTGTCT-3′; hNoxa Probe, 5′-TCAACTCAGGAGATTTG-3′ (5′-FAM/3′-BHQ1); hMCL-1 Fwd, 5′-GTTGACCAGAAAGGACACTCCAT-3′; hMCL-1 Rev, 5′-CAATCGTTTCCATATCAGTCAGAAA-3′; hMCL-1 Probe, 5′-TGTGAAACCGGCCTAAT-3′ (5′-FAM/3′-BHQ1); hA1 Fwd, 5′-CCTGGATCAGGTCCAAGCAA-3′; hA1 Rev, 5′-TTGGACTGAGAACGCAACATTT3′; hA1 Probe, 5′-TTGGACTGAGAACGCAACATTT-3′ (5′-FAM/3′-BHQ1). Real-time PCR results were normalized to endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression using the real-time primer/probe set Mouse GAPDH Endogenous Control (VIC/MGB Probe, Primer Limited; Applied Biosystems 435 2339) as per the manufacturer's instructions. All real-time PCRs were performed using the ABI Prism 7900HT and SDS2.1 software (Applied Biosystems).

Western Blot Analysis

Whole-cell extracts were prepared in cell lysis buffer (Cell Signaling Technology, Beverly, MA), and protein concentration was determined using Bio-Rad protein assay reagent as per the manufacturer's instructions. Protein (10 µg) was run out on 4% to 12% or 12% NuPAGE Bis Tris Gels (Invitrogen) and transferred to nitrocellulose membrane using the iBlot System (Invitrogen). Gels were blocked in 5% nonfat dry milk in tris-buffered saline with Tween 20 (TBST), and primary antibody was applied in 5% milk or BSA in TBST overnight at 4°C and washed five times for 5 minutes in TBST; secondary HRP-conjugated antibody was applied in 5% milk in TBST for 1 hour at room temperature and washed, and HRP visualization was performed using Western Lightening (Perkin Elmer, Waltham, MA) or Pico/Femto (Thermo Scientific, Pittsburgh, PA) chemiluminescent reagent. Antibodies were used at the following concentrations: 1:1000 rabbit antiPAX3 [26], 1:1000 rabbit anti-Noxa (ab36833; Abcam, Cambridge, MA), 1:1000 rabbit anti-Bmi-1, anti-Bim, anti-Bax, anti-Bak, anti-Bad, anti-Puma, anti-Bcl-2, anti-Bcl-xL, anti-Bcl-w (Cell Signaling Technology), 1:1000 anti-Bid (BD Pharmingen, Franklin Lakes, NJ), 1:4000, rabbit anti-MCL-1 (Sigma, St Louis, MO), 1:1000 rabbit anti-Pan-actin (Cell Signaling Technology), and goat anti-rabbit HRP (Jackson Laboratories, Bar Harbor, ME).

Caspase-3/7 Activity Assay

Primary myoblasts expressing PAX3-FOXO1 or empty vector were plated at 1000 to 2000 cells per well of a 96-well collagen coated plate (Becton Dickinson, Franklin Lakes, NJ). Twenty-four hours later, primary myoblasts were treated for 8 hours with indicated concentrations of GSI1 (Z-LLNle-CHO; Calbiochem, Darmstadt, Germany), bortezomib (LC Laboratories, Woburn, MA), or ABT-737 (Abbott Laboratories, Abbott Park, IL). ARMS cell lines were plated similarly and, 24 hours later, treated with drugs for 24 hours. Caspase-3/7 activity was determined using the Apo-ONE Homogeneous Caspase-3/7 Assay (Promega, Madison, WI) according to the manufacturer's instructions.

Cytochrome c Immunostaining

Cytochrome c staining was performed according to the protocol of Tait et al. [27]. Briefly, cells were plated on glass coverslips, and 24 hours later, cells were treated with 50 nM bortezomib for 24 hours. Then, cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature. Cells were then permeabilized with phosphate-buffered saline (PBS) and 0.1% Triton for 10 minutes. Cells were blocked for 30 minutes in PBS, 0.1% Triton X-100, and 0.5% BSA, followed by 1:200 anti-cytochrome c (BD Pharmingen; 556432) in blocking buffer overnight at 4°C. Cells were then washed five times in TBS and 0.05% Tween 20 and incubated with Alexa Fluor 488 goat anti-mouse antibody (Invitrogen) for 1 hour at room temperature. Samples were washed again and mounted in Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA), and fluorescent micrographs were taken.

In Vivo Bortezomib and ABT-737 Treatment

NOD-scid IL2Rγnull [28] mice were injected with 1 x 106 Arf-/- E7 PAX3-FOXO1 (30 animals total), PAX3 (5 animals), and empty vector (Empty) expressing (5 animals) primary myoblasts. Tumors were left to grow for 17 days at which time all mice had palpable tumors of at least 4-mm diameter. Mice were then treated twice a week for 2 to 4 weeks by tail vein injection with 0.8 mg/kg bortezomib (NCI/Millennium Pharmaceuticals, Cambridge, MA) in PBS, 75 mg/kg per day ABT-737 intraperitoneal (i.p.) in vehicle [30% propylene glycol, 5% Tween 80, and 65% dextrose (5%) in water], or 0.4 mg/kg bortezomib twice weekly intravenous (i.v.) and 75 mg/kg per day ABT-737 i.p. Per treatment, 15 animals were used for bortezomib only, 7 animals for ABT-737 only, and 6 animals for bortezomib and ABT-737 treatment. Tumors were monitored and measured daily with calipers, and mice were humanely killed once the tumor size reached 20% of their body weight and the tumor became ulcerated or interfered with animal mobility or function, adhering to the humane end points according to St Jude Institutional Animal Care and Use Committee (IACUC) protocol. Mice were monitored during treatment for signs of toxicity including weight loss, dehydration, and lethargy.

RH30 and RH41 xenografts were performed according to the Pediatric Preclinical Testing Program protocols as described by Houghton et al. [29] except that tumors were implanted subcutaneously in NOD-scid IL2Rγnull mice [28]. Six animals were used per treatment group and tumors were allowed to grow as above, before bortezomib treatment, and monitored as above.

Statistics

Statistical differences were determined by using a two-tailed t test, assuming equal variances. Survival curve differences were determined using the log-rank (Mantel-Cox) test in GraphPad Prism 5.02 software. P values are indicated by */‡P < .05, **P < .01, and ***P < .001.

Results

PAX3-FOXO1 Induces the Expression of Proapoptotic BH3-Only Protein Noxa in a p53-Independent Manner

Upon conducting microarray analysis of primary mouse myoblasts, expressing either empty vector, PAX3, or PAX3-FOXO1, Noxa was found to be specifically upregulated in PAX3-FOXO1-expressing cells (data not shown). This result was confirmed by real-time PCR comparing the expression of Noxa in primary myoblasts transduced with empty, PAX3, or PAX3-FOXO1 murine stem cell virus-internal ribosome entry site-green fluorescent protein (MSCV-IRES-GFP) retrovirus. This showed an increase in Noxa mRNA expression of 7.11 ± 1.63-fold (mean ± range, P < .001) in PAX3-FOXO1 over empty vector control cells (Figure 1A). The increase in Noxa expression in PAX3-FOXO1-expressing primary myoblasts can also be seen at the protein level (Figure 1B). Moreover, the mRNA expression of Noxa in PAX3-FOXO1 fusion-positive ARMS cell lines (RH30, RH4, RH41, RH3, RH28) was found to be upregulated 4-fold to 10-fold in all lines compared to Lonza HSMMs (Figure 1E). Given that Noxa is a known transcriptional target of p53 [30], we investigated whether the induction of Noxa by PAX3-FOXO1 was dependent on the p53 pathway by repeating the same experiment in Arf-/- and p53-/- primary myoblasts. The basal levels of Noxa expression are lower in Arf -/- and p53-/- primary myoblasts, likely because p53 transcriptionally activates Noxa expression [30]. PAX3-FOXO1 expression in these cell types continued to upregulate Noxa expression over empty vector control levels in the same cell type(Figure 1, C and D), indicating that PAX3-FOXO1 induction of Noxa expression is independent of p53 activity.

Figure 1.

Figure 1

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PAX3-FOXO1 induces Noxa expression in a p53-independent manner. (A) Relative mRNA expression of mouse Noxa determined by an average of two independently transduced primary myoblast samples by real-time PCR and normalized to GAPDH expression in primary myoblasts expressing PAX3, PAX3-FOXO1 (PF), or empty vector (Empty) and expressed as fold over empty vector control. (B) Western blots using anti-PAX3 antibody to detect PAX3 and PAX3-FOXO1, anti-Noxa antibody to detect Noxa, and anti- Pan-actin antibody as a loading control. Representative relative mRNA expression determined by real-time PCR comparing the expression of mouse Noxa in (C) Arf-/- and (D) p53-/- primary myoblasts and wild-type controls expressing PAX3, PAX3-FOXO1 (PF), or empty vector (Empty), normalized to GAPDH and expressed as fold over wild-type (WT) empty vector control; * indicates significance from appropriate empty vector control, and ‡ indicates significance from WT empty vector control. (E) Representative relative mRNA expression determined by real-time RT-PCR for human Noxa in ARMS cell lines RH30, RH4, RH41, RH3, and RH28 compared to HSMMs (Lonza) normalized to GAPDH and expressed as fold over HSMM control. (F) Representative relative expression of human MCL-1 mRNA determined by real-time PCR normalized to GAPDH expression, expressed as fold over HSMM control. (G) MCL-1 protein expression determined by Western blot; Pan-actin is provided as a loading control. (H) Representative relative expression of human A1 mRNA determined by real-time PCR normalized to GAPDH expression, expressed as fold over HSMM control.

We hypothesized that due to PAX3-FOXO1 up-regulation of Noxa, inhibition of Noxa activity might be required for ARMS tumorigenesis. The open reading frame of Noxa was sequenced and coded for wild-type Noxa protein in all ARMS lines and HSMMs (data not shown). Investigation of the mRNA expression levels of the Noxa antiapoptotic targets MCL-1 and A1 showed no correlation with Noxa expression (Figure 1, F and H). Because the levels of MCL-1 are also posttranslationally regulated by proteasome-dependent and proteasome-independent mechanisms [31,32], we examined the protein expression level of MCL-1 and A1. MCL-1 protein expression did differ from mRNA expression considerably; MCL-1 protein expression was upregulated in four of five ARMS lines: RH4, RH41, RH3, and RH28 over HSMMs (Figure 1G). This indicates that MCL-1 protein expression is increased through a posttranslational mechanism in these ARMS cell lines. A1 protein expression was below detection by Western blot (data not shown).

PAX3-FOXO1 Induction of Noxa Increases Myoblast Susceptibility to GSI1, Bortezomib, and ABT-737

Both GSI1 and bortezomib have been shown to induce p53-independent activation of Noxa resulting in apoptosis [19,20]. We tested if PAX3-FOXO1 induction of Noxa in primary mouse myoblasts would increase the susceptibility of these cells to undergo apoptosis upon treatment with these two drugs. In both wild-type and Arf-/- primary myoblasts, apoptosis was increased in a dose-dependent manner in cells expressing PAX3-FOXO1 compared to empty vector controls (Figure 2, A and B). By Western blot, both GSI1 and bortezomib treatment increased Noxa protein expression in primary myoblasts and, to a greater extent, in myoblasts expressing PAX3-FOXO1 (Figure 2C). Moreover, PAX3-FOXO1 expression reduced the protein expression of MCL-1 (Figure 2C) but not mRNA expression (Figure W1A). Increased Noxa expression can result in MCL-1 degradation [33]. This is likely the mechanism for PAX3-FOXO1 down-regulation of MCL-1 protein seen in primary myoblasts.

Figure 2.

Figure 2

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PAX3-FOXO1-expressing cells are more susceptible to apoptosis upon treatment with Z-LLNle-CHO (GSI1) or bortezomib. (A) Representative relative caspase activity of wild-type (WT) primary myoblasts expressing PAX3-FOXO1 (PF) or empty vector (Empty) control treated for 8 hours with increasing concentrations of GSI1 or bortezomib. Values are expressed as fold over untreated controls. (B) Representative relative caspase activity of Arf-/- primary myoblasts expressing PAX3-FOXO1 (PF) or empty vector (Puro) control treated for 8 hours with increasing concentrations of GSI1 or bortezomib. Values are expressed as fold over untreated controls. (C) Western blot of the WT primary myoblasts treated for 8 hours with vehicle alone or 2.5µM GSI1 or 50 nM bortezomib (Bort) for Noxa and MCL-1 expression. Pan-actin is used as a loading control. (D) Representative relative caspase activity of WT primary myoblasts expressing PAX3-FOXO1 (PF) or empty vector (Empty) control treated for 8 hours with increasing concentrations of ABT-737. (E) Relative Noxa mRNA expression in Arf-/- primary myoblasts expressing either a nontargeting (shNT) or Noxa-targeting (shNoxa) shRNA in control (Empty) and PAX3-FOXO1 myoblast lines. (F) Western blot for PAX3-FOXO1 and MCL-1 expression in Arf-/- control (Empty) and PAX3-FOXO1 primary myoblasts with nontargeting (shNT) or Noxa shRNA (shNoxa) knockdown. A Pan-actin loading control is provided. (G) Representative relative caspase activity in Arf-/- primary myoblasts expressing either a nontargeting (shNT) or Noxa-targeting (shNoxa) shRNA in control (Empty) and PAX3-FOXO1 myoblasts in the presence of 50 nM bortezomib. Values are expressed as fold over appropriate controls.

Due to the priming for apoptosis by Noxa induction, downstream of PAX3-FOXO1, we hypothesized that PAX3-FOXO1-expressing myoblasts may be more susceptible to compounds that induce apoptosis through inhibition of other antiapoptotic factors. The compound ABT-737 is known to selectively inhibit prosurvival factors Bcl-2, Bcl-xL, and Bcl-w but has no significant inhibitory effect on MCL-1 activity [34]. PAX3-FOXO1-expressing wild-type myoblasts were preferentially induced to undergo apoptosis in response to treatment with ABT-737 in a dose-dependent manner (Figure 2D). On the protein level, ABT-737 treatment of primary myoblasts resulted in a small increase in MCL-1 protein expression. However, MCL-1 protein is decreased in the presence of PAX3-FOXO1, and ABT-737 treatment results in a significant down-regulation of PAX3-FOXO1 and Noxa, indicating this increase in MCL-1 expression is partly downstream of PAX3-FOXO1 induction of Noxa (Figure W1, D–F).

To confirm that this increased propensity for bortezomib-induced apoptosis in PAX3-FOXO1-expressing myoblasts was Noxa dependent, we performed shRNA knockdown of Noxa expression in both empty vector and PAX3-FOXO1-expressing myoblasts and were able to reduce Noxa mRNA expression levels by about 65% (Figure 2E) in both empty vector and PAX3-FOXO1-expressing cells. shRNA knockdown of Noxa did not affect the expression level of PAX3FOXO1 in these cells (Figure 2F). However, Noxa shRNA expression did result in a partial rescue of MCL-1 expression levels (Figure 2F). Therefore, Noxa up-regulation contributes to the loss of MCL-1 protein expression seen with PAX3-FOXO1 expression (Figures 2, C and F, and W1D). The approximately 65% knockdown of Noxa was sufficient to completely abrogate the enhanced apoptosis seen in PAX3-FOXO1-expressing cells in the presence of 50 nM bortezomib (Figures 2G and W1B), indicating that this increased sensitivity of PAX3-FOXO1 to bortezomib-induced apoptosis is entirely due to Noxa up-regulation by PAX3-FOXO1. Similar abrogation of ABT-737-induced apoptosis was seen with Noxa shRNA expression (Figure W1C).

PAX3-FOXO1 Induction of Noxa Induces Apoptosis in Some, but Not All, ARMS Cell Lines

We also tested the sensitivity of the RH30, RH4, RH41, RH3, and RH28 ARMS cell lines to bortezomib and ABT-737, all of which express PAX3-FOXO1. ABT-737 induced apoptosis after 24 hours in all five ARMS cell lines (Figure 3A). Similar to primary myoblasts, ABT-737 upregulated MCL-1 concurrently with down-regulation of PAX3-FOXO1 (Figure W1E) though Noxa remained undetectable by Western blot (data not shown). RH30, RH41, and RH28 showed a significant apoptotic response after a 24-hour treatment with 50 nM bortezomib (Figure 3B), a concentration sufficient to cause apoptosis in primary myoblasts (Figure 2, A and B). Western blot analysis of these cells indicated that in response to treatment with bortezomib, all five cell lines upregulated Noxa protein expression (Figure 3C). MCL-1 protein expression was also upregulated by bortezomib in all cell lines, likely due to inhibition of MCL-1 proteasome degradation [16,22]. This increase in MCL-1 did not correlate with the survival seen in RH4 and RH3 and therefore does not account for the resistance of these cell lines to bortezomib treatment. Downstream of MCL-1, there is no mutation in direct pathway members (data not shown) nor is there a specific up-regulation of any prosurvival factor (BCL-2, BCL-xL, BCL-w) or down-regulation of any specific proapoptotic factor (BIM, BAX, BAK1, BAD, PUMA, tBID), which could account for the survival of RH4 or RH3 and the absence of cytochrome c release in these two surviving cell lines (Figure 3D). Moreover, upon bortezomib treatment, Noxa interacts with MCL-1 in both the susceptible RH30 and the resistant RH4 ARMS cell lines (Figure W1G). These cells are capable of undergoing apoptosis downstream of ABT-737 treatment, indicating that RH4 and RH3 have specifically lost the ability to undergo apoptosis in response to increased levels of Noxa, though the mechanism leading to this resistance remains unclear.

Figure 3.

Figure 3

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Apoptotic response of ARMS cell lines to ABT-737 and bortezomib treatment. Representative relative caspase activity of ARMS cell lines in response to treatment for 24 hours with increasing concentration of (A) ABT-737 and (B) bortezomib. (C) Western blots showing the protein expression of apoptotic pathway members in response to 50 nM bortezomib treatment in ARMS cell lines. Pan-actin is provided as a loading control. (D) Representative cytochrome c staining in ARMS cell lines treated with bortezomib. Arrows indicate cells where cytochrome c release has occurred and cytoplasmic cytochrome c can be detected.

Bortezomib Treatment In Vivo Reduces Tumor Growth

Arf-/- primary myoblasts (1 x 106) ectopically expressing E7 and PAX3-FOXO1 were injected subcutaneously into the hind flank of NOD-scid IL2Rγnull mice. Seventeen days later, all animals harbored palpable tumors of at least 4 mm in diameter and were treated twice weekly with 0.8 mg/kg per day bortezomib i.p. Animals lost a statistically significant amount of body mass following bortezomib treatment but regained weight 2 days after treatment (Figure 4A). Tumor dimensions were measured daily and animals treated with bortezomib showed a statistically significant reduction in tumor growth compared to PBS-treated controls (Figure 4, B and C); this was concurrent with a significant reduction in proportional tumor viability (Figure W2). Animals were sacrificed in accordance with animal welfare guidelines when the tumor reached 20% of the body mass (11/15 PBS, 12/15 bortezomib), the tumor became ulcerated (4/15 PBS), or because of tumor-induced paralysis not allowing the animal to reach food or water (1/15 Bort). In addition, 2/15 (13%) animals died due to bortezomib toxicity consistent with previously reported toxicity rates [22]. Comparing survival based on the day morbidity was reached, bortezomib-treated mice showed a small but significant increase in survival over vehicle alone (Figure 4D).

Figure 4.

Figure 4

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In vivo administration of bortezomib inhibits the growth of transformed primary myoblasts expressing PAX3-FOXO1. Fifteen animals per PAX3-FOXO1 treatment group and five animals each for empty vector (Empty) and PAX3 were used for i.v. bortezomib treatment. (A) Mouse body weight following treatment with 0.8 mg/kg twice weekly bortezomib i.v. (B) Fold change in tumor volume and (C) change in tumor volume in cm3 as calculated from tumor width and length measurements. (D) Survival of animals to morbidity from subcutaneous injection of cells. (E) Change in tumor volume in cm3 in seven mice per treatment group when treated with ABT-737 at 75 mg/kg per day i.p. (F) Change in tumor volume in cm3 in six mice per treatment group treated with ABT-737 at 75 mg/kg per day i.p. and 0.4 mg/kg twice weekly bortezomib i.v. Arrows indicate the days bortezomib was administered.

Treatment of this ARMS tumor model with ABT-737 at 75 mg/kg per day i.p. alone did not produce a large difference in tumor growth rate (Figure 4E) nor did it result in an increase in animal survival to morbidity (data not shown). We also wished to determine if a combination treatment of ABT-737 at 75 mg/kg per day i.p. and bortezomib at 0.4 mg/kg (half of concentration of bortezomib as a single agent) twice weekly i.v. could act synergistically in this model (Figure 4F). Initially, we found that the combination treatment appeared to work more effectively than 0.8 mg/kg twice weekly bortezomib i.v. treatment. However, the mice showed signs of accumulated toxicity by day 9, and treatment was ceased.

In addition, we tested the efficacy of bortezomib against RH30 and RH41 xenografts. RH28 xenografts have already been shown to respond when bortezomib was delivered i.p., but the RH30 and RH41 xenografts were unresponsive in this study [22]. However, in the clinic, bortezomib is administered i.v. and the NCI drug repository (personal communication) stated that bortezomib has poor bioavailability i.p. So we repeated the treatment of the same xenograft tumors using i.v. administration of bortezomib. RH41 showed a significant reduction in growth with bortezomib treatment i.v. (Figure 5, A and B), but RH30 did not respond (Figure 5, C and D), despite the induction of apoptosis in vitro (Figure 3A). Consistent with this result, the viable portion of tumor was reduced in RH41 treated with bortezomib but not RH30 (Figures W3 and W4).

Figure 5.

Figure 5

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In vivo administration of bortezomib inhibits the growth of ARMS tumor xenograft RH41 but not RH30. Six animals were used per tumor type and treatment group. (A) Change in tumor volume in cm3 as calculated from tumor width and length measurements following treatment of RH41 xenografts with 0.8 mg/kg twice weekly bortezomib i.v. (B) Survival to morbidity of mice post-RH41 xenograft. (C) Change in tumor volume in cm3 as calculated from tumor width and length measurements following treatment of RH30 xenografts with 0.8 mg/kg twice weekly bortezomib i.v. (D) Survival to morbidity of mice post-RH30 xenograft.

Discussion

We have shown that the ARMS-specific fusion gene PAX3-FOXO1 induces the expression of the proapoptotic BH3-only protein Noxa. Previously, it has been reported that expression of PAX3-FOXO1 induces apoptosis, and over time in culture, there is selection against high expressers of PAX3-FOXO1 [35]. We reasoned that if Noxa up-regulation limits the level of PAX3-FOXO1 expression, further increasing Noxa expression with drug treatment would induce apoptosis in PAX3-FOXO1-expressing cells at concentrations that do not affect controls. Indeed, this was the case. Both GSI1 and bortezomib are known to induce apoptosis in different cancer types through the p53-independent up-regulation of Noxa [16,19,20]. Like bortezomib, GSI1 can, in addition to inhibiting γ-secretase activity, inhibit proteasomal degradation indicating that the mechanism by which they induce apoptosis in myoblasts may be similar [16]. Primary mouse myoblasts expressing PAX3-FOXO1 are more susceptible to apoptosis induced by these drugs than empty vector counterparts. This is entirely due to PAX3-FOXO1 induction of Noxa expression as shRNA knockdown of Noxa completely abrogates this increase in apoptosis seen in PAX3-FOXO1-expressing primary myoblasts.

The BH3 mimetic compound ABT-737 specifically inhibits pro-survival factors Bcl-2, Bcl-xL, and Bcl-w in the nM range while showing low affinity for less homologous family members: MCL-1 and A1 [34]. High expression levels of MCL-1 have been shown to confer resistance to ABT-737, and sensitivity to ABT-737 is reestablished if MCL-1 levels are reduced [36]. The induction of Noxa expression allows an increase in association of Noxa with MCL-1, which inhibits MCL-1's antiapoptotic activity and can also promote proteasomal degradation of MCL-1 [37]. Consistent with this, we have found that PAX3-FOXO1-expressing myoblasts, which have increased Noxa and decreased MCL-1 protein expression, are more sensitive to ABT-737 treatment. ABT-737 treatment of both primary myoblasts and ARMS cell lines consistently resulted in up-regulation of MCL-1. This was concurrent with a down-regulation of PAX3-FOXO1 and Noxa (Figure W1, D and E). This is consistent with preferential apoptosis in high PAX3-FOXO1-expressing cells when treated with ABT-737, as the remaining cells express less Noxa and degrade less MCL-1 protein. Moreover, overexpression of MCL-1 allows for increased PAX3FOXO1 expression (Figure W1F). ABT-737 has been shown to synergize with bortezomib in apoptosis induction [38]. We also observed a synergistic effect when we used both drugs in an animal model of ARMS (Figure 4F), though we also noticed increased toxicity.

We have shown that PAX3-FOXO1 induces Noxa expression in a p53-independent manner and this is in concurrence with down-regulation of MCL-1 at the protein level. This up-regulation of Noxa may contribute to negative selection seen against high PAX3FOXO1-expressing cells [35]. Consistent with selection against high PAX3-FOXO1 expression in primary myoblasts, we have consistently been unable to overexpress PAX3-FOXO1 to the same level as other proteins in the same construct, such as PAX3 [10]. Moderate levels of sustained PAX3-FOXO1 expression induce a small seven-fold increase in Noxa expression, which alone is not sufficient to induce apoptosis. However, this level of Noxa expression does reduce prosurvival signals by decreasing the amount of MCL-1 protein (Figure 2C). This increases the sensitivity of PAX3-FOXO1-expressing cells to drugs such as bortezomib and ABT-737, which act to oppose prosurvival signaling.

Some of the ARMS tumor cell lines tested undergo apoptosis in response to bortezomib treatment. Previous studies using ARMS cell lines also showed an apoptotic response to bortezomib. Houghton et al. [22] found that RH30 and RH41 cell lines responded to bortezomib in vitro. However, when xenografts of the RH30, RH41, and RH28 tumors (from which the cell lines were derived) were treated with bortezomib, only RH28 showed inhibition. It should be noted that Houghton et al. [22] used a bortezomib dose of 1 mg/kg twice weekly and i.p. administration for which bortezomib has decreased bioavailability (personal communication from the NCI drug repository). We have shown that bortezomib is able to reduce the growth of RH41 xenografts if administered i.v. at 0.8 mg/kg twice weekly. RH30 xenografts continued to be nonresponsive to i.v. bortezomib treatment in vivo. Bersani et al. [21] showed that RH30 was responsive if bortezomib was administered intratumorally at 1.25 mg/kg twice weekly, which indicates that higher doses are required to induce apoptosis in RH30 in vivo. Unfortunately intratumoral treatment is not feasible in humans, particularly those with disseminated disease, and systemic treatment with such high doses is also not feasible as it would result in unacceptable toxicity due to the steep dose-response curve of bortezomib [22]. Therefore, bortezomib may not be the most appropriate drug with which to target this proapoptotic pathway in ARMS. Bortezomib functions as a proteasome inhibitor and thus has numerous other effects on cell biology in addition to Noxa up-regulation. If a more targeted drug that specifically induced Noxa expression or mimicked the function of Noxa was developed, we would predict that this would be a more efficacious molecule than bortezomib, for the specific induction of apoptosis in PAX3-FOXO1-expressing cells. Here, we have established that Noxa up-regulation downstream of PAX3-FOXO1 occurs in ARMS cells and sensitizes these cells to apoptosis upon treatment with drugs that inhibit prosurvival signals. How best to exploit this pathway for the treatment of human ARMS remains to be determined.

Not all ARMS cell lines induced apoptosis in response to bortezomib treatment. In comparison to our primary myoblast model, where PAX3-FOXO1 expression results in a reduction of MCL-1 at the protein level, in ARMS cell lines MCL-1 protein is increased in four of five lines compared to HSMMs. This does not correlate well with mRNA expression, indicating that MCL-1 protein stability is enhanced in some ARMS cell lines. Moreover, two of five ARMS cell lines are resistant to bortezomib even at 10x the dose required to kill the other three of five cell lines and mouse primary myoblasts expressing PAX3-FOXO1. Bortezomib resistance in cancer cells can be conferred by mutation or up-regulation of proteasome machinery or up-regulation of drug efflux pumps [39]. However, this is apparently not the case in ARMS cell lines as bortezomib treatment causes a similar increase in Noxa expression in both bortezomib-sensitive and bortezomib-resistant ARMS cell lines. This indicates that bortezomib inhibits the proteasome in all ARMS cells and causes Noxa up-regulation through the unfolded protein response [11]. Therefore, we looked downstream of Noxa in the apoptosis pathway. No association was found between the expression of members of the mitochondrial apoptotic pathway and bortezomib resistance (Figure 3C). Moreover, none of the proteins thought to mediate the Noxa proapoptotic signal were mutated to prevent apoptosis signaling. All ARMS cell lines are capable of undergoing caspase-dependent apoptosis as ABT-737 produced an apoptotic response in all five ARMS cell lines.

Though we were unable to determine the mechanism by which ARMS cell lines RH4 and RH3 become resistant to Noxa induction by bortezomib, the fact that they are resistant may indicate an important aspect of ARMS tumor biology. ARMS tumors appear to posttranslationally increase MCL-1 protein expression compared to HSMMs and, in some cases, can also become completely resistant to Noxa induction by bortezomib. This suggests that ARMS tumor cell lines develop the means to adapt to increased Noxa expression and, in some cases, completely overcome its proapoptotic effects. We envisage that a similar path to resistance could develop in ARMS tumors.

In this study, we have identified Noxa as a protein specifically upregulated downstream of PAX3-FOXO1 expression in primary myoblasts. This up-regulation of a proapoptotic protein by PAX3FOXO1 results in an increased sensitivity of cells to anticancer drugs such as GSI1, bortezomib, and ABT-737. Therefore, this study demonstrates that this ARMS-specific Noxa-driven apoptosis pathway represents a novel target for specific therapy against PAX3-FOXO1- expressing ARMS tumor cells.

Supplementary Material

Supplementary Figures and Tables
neo1507_0738SD1.pdf (1.6MB, pdf)

Acknowledgments

The authors thank the NCI drug repository for supply of the bortezomib compound and Abbott for the supply of ABT-737. The authors also thank Joseph Opferman, Stephen Tait, and Chris Morton for their technical advice and Colin Pritchard and Brendan Hobbs who originally identified Noxa as a target of PAX3-FOXO1 by microarray.

Footnotes

1

This work was supported by grant funding from the Van Vleet Foundation of Memphis and by the American Lebanese Syrian Associated Charities of St Jude Children's Research Hospital. The authors do not have any competing financial interests in relation of the work described.

2

This article refers to supplementary materials, which are designated by Figures W1 to W4 and are available online at www.neoplasia.com.

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Associated Data

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Supplementary Materials

Supplementary Figures and Tables
neo1507_0738SD1.pdf (1.6MB, pdf)

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