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Experimental Therapeutics, Molecular Targets, and Chemical Biology |
Departments of 1 Dermatology, 2 Chemistry, and 3 Obstetrics and Gynecology, University of Michigan Comprehensive Cancer Center, Ann Arbor, Michigan; 4 Millennium Pharmaceuticals, Inc., Cambridge, Massachusetts; and 5 Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Requests for reprints: María S. Soengas, University of Michigan Comprehensive Cancer Center (4217 CCGC), 1500 East Medical Center Drive, Ann Arbor, MI 48109. Phone: 734-936-5643; Fax: 734-647-9654; E-mail: soengas{at}umich.edu.
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Abstract |
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B, Bcl-2, Bcl-xL, XIAP, TRAF-2, or FLIP). Instead, we identified a dramatic induction in vitro and in vivo of the BH3-only protein Noxa in melanoma cells (but not in normal melanocytes) in response to proteasome inhibition. RNA interference validated a critical role of Noxa for the cytotoxic effect of bortezomib. Notably, the proteasome-dependent regulation of Noxa was found to extend to other tumor types, and it could not be recapitulated by standard chemotherapeutic drugs. In summary, our results revealed Noxa as a new biomarker to gauge the efficacy of bortezomib specifically in tumor cells, and provide a new strategy to overcome tumor chemoresistance. |
Introduction |
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The rational design of improved therapeutics has been complicated by the complexity of genetic alterations acquired during melanoma progression (2). Hyperactivation of efflux pumps, detoxification enzymes, and a multifactorial alteration of survival and apoptotic pathways have been proposed to mediate the multidrug-resistant (MDR) phenotype of melanoma cells (3, 4). However, no factor has been consistently identified as a gold-standard marker for melanoma progression or melanoma maintenance.
Agents with pleiotropic effects that are able to interfere simultaneously with the expression (and function) of survival and apoptotic factors may offer an alternative to standard single-target drugs. In this context, the proteasome inhibitor bortezomib (Velcade, previously known as PS-341) is raising great enthusiasm as a new class of anticancer agent that is not subject to classic MDR-dependent inactivation (8, 9). Bortezomib has a wide spectrum of action in hematologic and solid tumors, including lung, breast, prostate, pancreatic, and head and neck carcinomas (9, 10). Based on its efficacy, the FDA has approved the use of bortezomib for the treatment of refractory multiple myeloma (11), and several clinical trials are under way testing its therapeutic value in a variety of cancers (12, 13).
Interestingly, intratumoral injections of bortezomib have been recently reported to reduce the localized growth of human melanoma xenografts in mice, particularly when combined with the DTIC derivative temozolomide (14). Important questions remain as to the mechanistic basis underlying the higher sensitivity of melanoma cells than normal melanocytes to bortezomib. In fact, the proteasome controls the half-life of the vast majority of cellular proteins (15), and it has been rather puzzling that proteasome inhibitors such as bortezomib can be clinically effective while displaying an acceptable safety profile. Thus, although bortezomib is generally considered a potent inducer of cell death, the mechanism(s) underlying its tumor cell selectivity is poorly understood (even in tumor types where bortezomib is being tested in clinical settings; ref. 9). Up-regulation of proapoptotic factors such as Bax (16) and down-regulation of the antiapoptotic proteins Bcl-2, Bcl-xL, XIAP, and FLIP, in part through the inactivation of the nuclear factor-B (NF-B) pathway, have been proposed as frequent facilitators of cell death in bortezomib-treated tumor cells (17–22). However, whether these changes in expression are cause or consequence of the death process and whether they are general mediators of the cytotoxic effect of bortezomib is unclear (9).
Here we report on a comprehensive analysis of the effect of bortezomib on the apoptotic machinery of normal melanocytes and a panel of 23 melanoma lines. Standard chemotherapeutic agents with different cellular targets (Adriamycin and cisplatin) were analyzed in parallel to reveal events that may be uniquely activated by bortezomib in tumor cells. We identify a novel mechanism of action of bortezomib in melanoma and other tumor cells that depends on the induction of the proapoptotic protein Noxa. This work is an important step in understanding how melanoma chemoresistance can be overcome by blocking protective signals dependent on the proteasome that maintain the apoptotic machinery of melanoma cells in a dormant state.
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Materials and Methods |
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Reagents. Bortezomib (Velcade, formerly PS-341) was obtained from Millennium Pharmaceuticals (Cambridge, MA). For analyses of cell death and cell cycle in tissue culture systems, bortezomib was reconstituted in DMSO at a concentration of 0.1 mmol/L; for studies in vivo, it was prepared in a 0.85% (w/v) sterile saline solution. Adriamycin (doxorubicin) and cisplatin were obtained from Sigma Chemical (St Louis, MO).
Cell viability assays. The percentage of death cells at the indicated times and drug concentrations was estimated by standard trypan blue exclusion or 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assays as described elsewhere (25). For simplicity, control samples, which were incubated only with solvent (0.05-0.1% DMSO), are indicated as nontreated. Analysis of chromatin condensation and cellular ultrastructure by electron microscopy are indicated in the Supplementary Information.
Cell cycle analyses. Normal melanocytes or melanoma cells (5 x 105) were treated with bortezomib (50 nmol/L) or vehicle control. Cell cycle analyses were conducted after a 2-hour incubation of cells in labeling solution (50 µg/mL of propidium iodide (PI; Sigma Chemical), in PBS containing 0.2% Triton and 10 µg/mL RNase A. PI fluorescence was measured using a FACSCalibur. Cell cycle distribution was determined with a Cell Quest software (Becton Dickinson, San Jose, CA).
Protein immunoblots. To determine changes in protein levels, 2 x 106 cells were treated with Adriamycin (0.5 µg/mL), bortezomib (50 nmol/L), or cisplatin (30 µg/mL) and harvested at the indicated time points. Total cell lysates were subjected to electrophoresis in 12%, 15%, or 4% to 15% gradient SDS gels under reducing conditions, and subsequently transferred to Immobilon-P membranes (Millipore, Bedford, MA). Protein bands were detected by enhanced chemiluminescence system (see Supplementary Information for a list of the antibodies used in this study). Cytosolic and membrane-rich fractions were prepared by digitonin extraction essentially as previously described (26). Cytosolic fractions (100 µg) and 25 µg of mitochondrial fractions were used to visualize Cyt c, Smac, HtrA2, and AIF release by Western blotting. Control antibodies were COX IV for the mitochondrial fraction and ß-actin for the cytosolic fraction.
Fluorometric caspase activity assays. Protein (20 µg) of cytosolic fraction from the digitonin fractionation were incubated with 200 µL of Activation Buffer [50 mmol/L Pipes (pH 6.85), 0.1 mmol/L EDTA, 10%glycerol, 1 mmol/L DTT] in the presence of 20 µmol/L of one of the following 7-amino-4-trifluoromethyl coumarin (AFC) derivatives: Ac-LEHD (preferentially cleaved by Casp-9, Casp-5, and related cystein proteases), Ac-IETD (as a preferred substrate of Casp-8, Casp-6), or Ac-DEVD (preferentially cleaved by Casp-3, Casp-7, and related caspases). Proteolytic release of AFC was monitored as indicated by the provider (Biomol Research, Plymouth Meeting, PA) at a 
exc = 400 nm and em = 505 nm using a CytoFluor Multiwell Plate Reader Series 4000 (Applied Biosystems, Framingham, MA). Signal was expressed as arbitrary fluorescent units (AFU)/min. Experiments were done in triplicate.
Stable RNA interference. To down-regulate the expression of Noxa by RNA interference (RNAi), oligonucleotides allowing for the generation of 19-bp short hairpin RNAs (shRNA) were designed following indications by the OligoRetriever Database (http://katahdin.cshl.org:9331/RNAi_web/scripts/main2.pl). BLAST search was done to ensure at least 4-nucleotide (nt) differences with annotated human genes. The corresponding oligonucleotides were annealed and cloned under the control of the H1 promoter into a self-inactivating lentiviral vector (27). The vector was also designed to carry the green fluorescent protein reporter gene under control of the human ubiquitin-C promoter to monitor infection efficiency. Lentiviral infections were done essentially as described elsewhere (27) and the potency and specificity of each construct was determined by protein immunoblotting (see text). The constructs generated were as follows: Noxa (1), nt 1177 to 1195 and Noxa (2), nt 295 to 313. Scrambled oligonucleotides were also designed to generate control shRNA. Cloning strategies and primer sequences are available from the authors on request.
Quantitative real-time PCR. Total RNA from cultured cells was obtained by using the RNeasy Mini Kit (Qiagen, Inc., Chatsworth, CA). Total RNA (0.5 µg) was reverse-transcribed using the Superscript III reverse transcription kit (Invitrogen, Carlsbad, CA). Relative mRNA levels of Noxa and the housekeeping gene ß-actin were determined by reverse transcription-PCR (RT-PCR) using a LightCycler DNA Master SYBR Green I kit (Roche Diagnostics GmbH, Mannheim, Germany) and an automated PCR station (BioMek 2000, Beckman-Coulter, Miami, FL). Typical amplification reactions (15 µL) contained 2.5 mmol/L MgCl2, 0.6 µmol/L of a primer mix, 1.5 µL of SYBR Green Mix, and one tenth of the reverse transcription reaction. A 7700 Sequence Detector (Applied Biosystems) was programmed for an initial step of 5 minutes at 95°C followed by 35 cycles of 30 seconds at 95°C, 30 seconds at 60°C, and 30 seconds at 72°C. The oligonucleotides used as specific primers for Noxa were 5'-ATGAATGCACCTTCACATTCCTCT (sense) and 5'-TCCAGCAGAGCTGGAAGTCGAGTGT (antisense). Fluorescence was automatically monitored during every PCR cycle and during the post-PCR temperature ramp. For each sample, RNA content was normalized to ß-actin. Fluorescence values were expressed with respect to the basal expression of nontreated normal melanocytes. The specificity of PCR amplification of each primer pair was confirmed by analyzing PCR products by agarose gel electrophoresis and by melting curve analysis.
Melanoma growth in vivo (mouse xenografts). Female athymic nude mice (Taconic Farms, Germantown, NY) were kept in pathogen-free conditions and used at 8 to 12 weeks of age. Animal care was provided in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals of the University of Michigan. To analyze localized growth of melanoma cells in vivo, 0.5 x 106 melanoma cells were injected s.c. in both rear flanks (n = 10 tumors per experimental condition). Treatment was initiated 48 hours after tumor implantation. Bortezomib was given systemically by i.p. injection (1.0-1.3 mg/kg) in a rotating 2-day schedule. Similar results were obtained for treatments done via i.v. administration (data not shown). Animals were weighed every 2 days to ensure maintenance of total weight within 80% of control populations. Tumor volume was estimated as V = L x W2 / 2, where L and W stand for tumor length and width, respectively. Tumors were collected at day 17 postinjection and snap frozen; 0.4-µm sections were prepared for histologic analyses to guide with manual microdissection (to restrict tissue specimens to areas with >70% tumor cellularity) for subsequent protein isolation.
Statistics. Statistical analysis of tumor growth in vivo was done using the Statistical Package of the Social Sciences (SPSS) version 11.5 for Windows. Nonparametric Mann-Whitney U test was used for two-group comparisons. Two-tailed P < 0.05 was considered statistically significant.
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Results |
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Adriamycin was able to kill the "early-stage" radial growth phase cells, but most vertical growth phase and metastatic lines were resistant (see relative death rates in Fig. 1A and dose-response curves for representative examples of metastatic lines in Fig. 1B, left). In contrast, all lines died after bortezomib treatment (Fig. 1A-B). These include aggressive lines such as, for example, lines 9 and 10, expressing low levels of Apaf-1 and high levels of antiapoptotic factors such as Bcl-2, Bcl-xL, Mcl-1, or Survivin (see Supplementary Table 1). Similarly, melanoma-associated mutations in p53, Ras, B-Raf, or the INK4a/ARF locus did not compromise the response to bortezomib (Fig. 1A; Supplementary Table S1). The tumor cell–selective effect of bortezomib was illustrated by a death rate below 30% of normal melanocytes (even at doses of 100 nmol/L, 10 times the EC50 for melanoma cells; Fig. 1B, middle). Interestingly, analysis of cell cycle progression by standard flow cytometry assays indicated that melanocytes did in fact respond to bortezomib and stop proliferating at the G2-M phases of the cycle (Fig. 1C, left). However, whereas normal melanocytes and remained viable, melanoma cells seemed to have fragmented DNA (measured as a sub-G0 population; Fig. 1C).
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Activation of extrinsic and intrinsic apoptotic pathways by bortezomib. 4',6-Diamidino-2-phenylindole (DAPI) staining and electron microscopy revealed classic apoptotic hallmarks in bortezomib-treated melanoma cells (such as chromatin condensation, membrane blebbing, and maintenance of the integrity of cellular membranes; see Supplementary Fig. S1). Therefore, we focused on apoptotic pathways to address the mechanism of action of bortezomib and to determine quantitative and/or qualitative differences with Adriamycin and cisplatin.
Caspase processing and activation were assessed in five representative cell lines. Metastatic lines 3 and 5 are sensitive to DNA-damaging agents and process regulatory (Casp-9 and Casp-8) and effector caspases (Casp-3 and Casp-7) after Adriamycin treatment (Fig. 2A). The vertical growth phase line V4 and the metastatic lines 9 and 10 are more resistant to Adriamycin, and as previously described (24) have an impaired caspase activity (see Fig. 2A). Interestingly, all lines cleaved regulatory and effector caspases in response to bortezomib (Fig. 2A). Standard fluorescence-based proteolytic assays indicated that caspases processed in response to bortezomib were in fact functional (Fig. 2B). The response to cisplatin, however, was more heterogeneous. Thus, cisplatin-induced cell death did not necessarily correlate with caspase processing. Note for example an 80% killing with a barely detectable processing of Casp-9, Casp-8, and Casp-3 in cisplatin-treated lines 3 or 10 (Fig. 2A, lanes 4 and 16), respectively).
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Changes in the levels of apoptotic factors frequently associated with the nuclear factor-B pathway do not precede Cyt c release nor caspase activation. Time courses were done to monitor the release of death inducers from the mitochondria and identify early events involved in the activation of apoptotic pathways. To this end, enriched cytosolic and membrane-enriched fractions were prepared from control untreated and bortezomib-treated cells and changes in protein expression were determined as a function of time (see Fig. 3 for representative examples of metastatic melanoma lines 3 and 9). Using this strategy, the release of Cyt c, Smac, and HtrA2 was detectable 12 to 18 hours after treatment (Fig. 3A).
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Inhibition of the NF-B has also been shown to favor bortezomib-induced caspase in some, although not all, tumor types (9, 10). Reporter plasmids containing canonical NF-B binding sites showed no significant inhibition of the intrinsic basal transcriptional activity of NF-B by bortezomib in the melanoma cells tested in this study (see Supplementary Fig. S3). Consistent with these results, the total amount of IB, a NF-B inhibitor, did not significantly change during bortezomib treatment (Fig. 3C). Proteasome inhibition however could in fact block the exogenous activation of NF-B (e.g., by tumor necrosis factor-
, TNF-; see Supplementary Fig. S3).
With respect to proapoptotic factors, no consistent up-regulation was found for Bax or Bak by bortezomib (Fig. 3C). Moreover, cell death was not favored by an increase of Apaf-1 levels; in fact, this protein was cleaved during treatment (Fig. 3C). Bim was found to be induced by bortezomib (Fig. 3C). However, this protein was also up-regulated by bortezomib in normal melanocytes, which remained viable (see Supplementary Fig. S4).
An alternative mechanism that could account for Cyt c is the activation of the death receptor pathway via Casp-8-mediated Bid cleavage (34). However, Bid processing was detected substantially after the release of Cyt c (Fig. 3A and C). Therefore, it is also unlikely that Bid is an initial trigger of mitochondrial dysfunction in bortezomib-induced melanoma death.
In summary, bortezomib-mediated release of mitochondrial death inducers is not preceded in melanoma cells by a significant cleavage of Bid nor a NF-B-dependent down-regulation of Bcl-2, Bcl-xL, XIAP, FLIP, or TRAF-2, all previously associated with melanoma chemoresistance (3, 24).
Stress-associated signals are not early drivers of the selectivity of bortezomib towards melanoma cells. A regulated release of Cyt c can be also be favored by the activation of a series of stress-related signals that can be controlled by the proteasome. These include the unfolded protein response (UPR) at the endoplasmic reticulum and the induction of stress kinases such as p38 and c-jun NH2-terminal kinase (JNK; ref. 10). In melanoma cells, the classic UPR proapoptotic factor CHOP and the chaperone Grp-78 can be induced by bortezomib (Fig. 4A). However, the up-regulation of CHOP was found also in normal melanocytes, and did not correlate with the extent of cell death. The induction of Grp-78 was detectable at late time points after bortezomib treatment (Fig. 4A). Similarly, melanoma cells are not dependent on JNK or p38 function to respond to bortezomib by inducing apoptosis. Pharmacologic inhibitors of p38 (PD-169316, SB-220025, SB-239063, and SC-68376) and JNK kinases (SP-600125, D-JNK peptide inhibitor 1, or L-JNK peptide inhibitor 1) were unable to protect melanoma cells from bortezomib's killing. Moreover, these inhibitors increased the toxicity of bortezomib on normal melanocytes.
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To determine whether induction of Noxa by bortezomib is a general event in melanoma cells, a total of 16 metastatic melanoma lines independently isolated and with different genetic backgrounds (24, 35) were analyzed. As shown in Supplementary Fig. S5, 15 of these 16 lines accumulated Noxa at early time points after treatment. Intriguingly, despite Noxa being reported as a p53 target (36, 37), the accumulation of Noxa by bortezomib is likely p53-independent because it was also observed in melanoma lines such as line 4 (SK-Mel-28), expressing the inactive R273H p53 mutant. Defects in the p53 regulator p14ARF (e.g., lines 10, 15, or 17) did not compromise Noxa up-regulation (see Supplementary Fig. S5). Of note, Noxa has been described as able to promote the release of Cyt c without inducing large-range swelling of isolated mitochondria (38), and p53-independent induction of Noxa has been previously reported in melanoma cells (39).
Drug-selective activation of Noxa by bortezomib. Parallel treatments of melanoma lines 3 and 9 with Adriamycin, cisplatin, and bortezomib showed a drastic difference in the effect on these different drugs on Noxa expression. Compare for example the 2- to 3-fold induction of Noxa by Adriamycin or cisplatin (visualized after long exposures of protein immunoblots) with >75-fold induction by bortezomib (Fig. 4B). Supporting a p53-independent regulation of Noxa in melanoma cells, the levels of Noxa and p53 showed no directly correlation. As shown in Fig. 4B, the accumulation of p53 was higher in lines treated with Adriamycin and cisplatin than with bortezomib.
Transcriptional up-regulation of Noxa by bortezomib. The accumulation of Noxa by bortezomib could be a simple response of blockage of protein degradation or involve activation of gene expression. To this end, quantitative RT-PCR (see Materials and Methods) was done in normal melanocytes and melanoma lines 3 and 9, at time t = 0, 1, 3, 6, 12, and 24 hours after bortezomib treatment. ß-actin was used as control housekeeping gene whose expression is not modulated by bortezomib (see Fig. 3A). As shown in Fig. 5A, the intrinsic levels of Noxa mRNA in melanoma cells (t = 0 hour) were found to be 2- to 6-fold higher than in normal melanocytes, with the highest expression in the most sensitive line 9. Bortezomib treatment induced a further accumulation of Noxa mRNA more effectively in the tumor cells than normal melanocytes. For example, whereas at 12 hours posttreatment a 4-fold increase of mRNA levels was detected in normal melanocytes, melanoma lines 3 and 9 expressed 42- and 57-fold higher Noxa mRNA than the untreated normal controls (Fig. 5A). Therefore, the tumor-selective up-regulation of Noxa may rely on higher endogenous mRNA levels in melanoma cells than normal melanocytes, further augmented during treatment by new mRNA and protein synthesis.
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Bortezomib-mediated up-regulation of Noxa in vivo. Altogether, our results support Noxa expression as a surrogate to address early events driven by bortezomib in melanoma cells. To confirm this hypothesis, the activation of Noxa was analyzed in vivo, by monitoring changes in gene expression in melanoma cells grown as mouse xenografts. Of the panel of cell lines shown in Fig. 1, lines 9 and 10 were chosen on the basis of displaying the most aggressive growth after s.c. implantation in immunosuppressed mice (data not shown). Bortezomib has been recently shown to block melanoma growth in mice when injected peritumorally (14). To better recapitulate administration routes used in the clinic, mice were treated systemically with bortezomib or placebo control as indicated in Materials and Methods. Although bortezomib will have to be potentiated with additional chemotherapeutic agents, it noticeably reduced tumor growth (see Fig. 6A, a-b; Supplementary Fig. S6). Histologic evaluation of untreated and bortezomib-treated xenografts (Fig 6A, c-f) showed a noticeable reduction of mitotic figures and changes in the tumor architecture (with increased fibrotic areas and tumor cell collapse). More importantly, protein extracts prepared from the tumor xenografts revealed a clear induction of Noxa by bortezomib in vivo (Fig. 6B). Therefore, our results provide the proof of concept for the use of Noxa as biomarker to address proteasome inhibition in tumor cells.
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Discussion |
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Unexpected mechanism underlying the efficacy and selectivity of bortezomib. The proteasome controls the half-life of the vast majority of cellular proteins. Therefore, identifying the main drivers of bortezomib's toxicity within a myriad of inconsequential byproducts of proteasome inhibition has been a major challenge in all tumor types where this drug has been tested (9, 10). Consequently, it is not surprising that gold-standard markers of early events induced by bortezomib in a tumor-selective manner have remained elusive (9).
Perhaps one of the most intriguing results of this study is that bortezomib was able to promote an effective release of Cyt c and other mitochondrial death inducers without down-regulating protective signals dependent on Bcl-2, Bcl-xL, XIAP, TRAF-2, or FLIP, frequently associated with melanoma chemoresistance. Therefore, and in contrast to multiple myeloma, lymphoma, Hodgkin disease, or pancreatic or lung cancer cells (18, 19, 40–42), our data is not consistent with a proteasome/IB-dependent regulation of the above indicated antiapoptotic factors. In addition, reporter plasmids containing canonical NF-B binding sites indicated that although bortezomib was able to block exogenous activation for example by TNF-, its effects on the basal transcriptional activity of NF-B were not statistically significant. It should be noted that our functional analyses of NF-B activation (Supplementary Fig. S3) are compatible with those of Amiri et al. (14). These authors found a limited effect of bortezomib on the intrinsic levels of the NF-B targets such as CXCL8 or MDR-1, but a compensatory effect upon Temozolomide treatment. Our results however do not support the hypothesis of caspase activation driven by the down-regulation of antiapoptotic factors controlled by NF-B.
In addition, a comprehensive analysis of the effect of bortezomib the dissipation of the mitochondrial membrane permeability, induction of JNK- and p38-dependent stress pathways did not support these events as initiating or rate-limiting drivers of the selective effect of bortezomib. In marked contrast, Noxa was identified for the fist time as an early gene specifically restricted to tumor cells and whose inactivation by RNAi significantly affected the kinetics and dose-response of bortezomib.
The apoptotic machinery in melanoma cells: kept in a dormant state? Melanoma has been invariably linked to defective apoptotic pathways and multidrug resistance (4). However, the similar sensitivities shown here for bortezomib on a large panel of cell lines independently isolated from early, intermediate, and late stages of the disease, clearly indicate that despite multiple genetic and epigenetic defects accumulated during tumor progression, melanoma cells still retain the core machinery to engage the activation of the apoptotic caspases. An important corollary of this study is that neither high levels of antiapopotic Bcl-2 family members, nor classic melanoma-associated mutations in N-Ras, B-Raf, p53, and the INK4a/ARF locus, as well as increased levels of Survivin (see Supplementary Table S1) can prevent melanoma cell death if the proteasome is blocked. Similarly, although low Apaf-1 levels can compromise the response of melanoma cells to Adriamycin (24, 43), Paclitaxel (44), and high doses of etoposide (45), this is not the case for bortezomib. Because none of the lines in this study is completely deficient for Apaf-1 (see Table 1 and ref. 24), it is conceivable that the high levels of cytosolic Cyt c and Smac released by bortezomib in response to Noxa compensate for reduced Apaf-1 expression and/or high levels of antiapoptotic signals that may prevent caspase activation (e.g.,high levels of XIAP or Ml-IAP). The release of AIF and of HtrA2 from the mitochondria could further contribute to caspase-independent death events, particularly if caspases are inactivated (Supplementary Fig. S2). It should be noted that cisplatin can also bypass low Apaf-1 expression (e.g., in lines 9 or 10). Nevertheless, at the doses required to activate the death machinery, cisplatin is also toxic for normal melanocytes and thus not selective.
At face value, the dramatic up-regulation of Noxa in response to proteasome inhibition, and the subsequent release of proapoptotic factors from the mitochondria underscore a critical role of the proteasome in maintaining apoptotic programs in a latent or suppressed state. In this context, melanoma cells may be more dependent on proteasomal degradation than normal melanocytes because they need to repress their intrinsically high levels of Noxa mRNA. It could be argued that oncogenic transformation drives the activation of a proteasome-controlled transcription factor able to transactivate Noxa and/or stabilize its mRNA. In keeping with this view, and as described for proapoptotic functions of c-Myc or E2F-1 (46), the accumulation of Noxa as a "byproduct" of tumor development may serve as the Achilles' heel for the selective destruction of malignant cells.
Noxa as a biomarker of proteasome inhibition. Given the pleiotropic nature of the proteasome, it would not be expected that Noxa is the sole initiator of the cytotoxic effect of bortezomib. Thus, we have shown both by flow cytometry in cultured cells and by histologic analyses in tissue specimens, that bortezomib can effectively interfere with cell cycle progression. Still, and precisely because the large fraction of proteins that could have been affected by this inhibitor, a 50% reduction in cell death observed by RNAi against a single gene (Noxa) is highly significant. Moreover, our results could have potential clinical implications. As indicated before, bortezomib is being actively tested in clinical trials against multiple tumor types, but no marker has been consistently identified to address drug efficacy specifically in tumor cells. Here we showed a generalized accumulation of Noxa in 15 of 16 metastatic melanoma lines tested, as well as in cell lines from T-cell leukemia, breast, and non–small cell lung cancer (Jurkat, MDA-MB-321, and H460, respectively). Therefore, based on the dramatic difference in expression between tumor and normal cells (>50-fold induction), we propose Noxa as a novel biomarker to address proteasome inhibition specifically in tumor cells. In addition, we have shown the induction of Noxa in archived frozen specimens from tumor xenografts treated in vivo. Therefore, the possibility of analyzing Noxa by immunologic approaches may facilitate a systematic analysis of tumor-specific proteasome inhibition on biopsies collected pretreatment and posttreatment. Furthermore, the fact that Noxa can be up-regulated in p53 mutant lines [e.g., SK-Mel-28 (here referred as melanoma 4), Jurkat, or MDA-MB-231] support the use of this BH3-only protein as a surrogate for proteasome inhibition even in p53-defective tumor types.
In summary, this study has revealed a novel mechanism to activate the latent apoptotic machinery of melanoma cells that cannot be recapitulated by Adriamycin and cisplatin, as examples of two drugs with different modes of action. Moreover, bortezomib has served as powerful experimental tool to address the interplay between the proteasome and proapoptotic and antiapoptotic factors acting upstream, downstream and at the level of the mitochondria. Our results identify Noxa as a new marker to assess the effect of bortezomib in vitro and in vivo and provide the basis for the rational design of novel therapeutics to overcome tumor chemoresistance by exploiting intrinsic differences in the requirement of the proteasome for the maintenance of the viability of normal and tumor cells.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Rebecca Mosher, Sarah Waywell (Millennium Pharmaceuticals), Mila M. McCurrach, and Sagarika Ray (Cold Spring Harbor Laboratory) for help and advice with mouse xenografts at an early phase of this work; the following investigators at the University of Michigan: Laure Rittie for the analysis of gene expression by quantitative RT-PCR, James T. Elder and Rajan Nair for sequencing of B-Raf and N-Ras, and Audrey Bengston and Wen-Hua Tang for technical assistance; José Esteban, Gabriel Núñez, Mikhail Nikiforov, and Andrzej A. Dlugosz for helpful suggestions and critical reading of this article; Colin S. Duckett's laboratory for reagents and advice; and Brian Nickoloff (Loyola University) for sharing data before publication regarding the regulation of Noxa by bortezomib.
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Footnotes |
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P. Steiner is currently at the ImClone Systems, Inc., New York.
6 Y.F. and M.S.S., unpublished results. 
Received 2/28/05. Revised 4/11/05. Accepted 4/20/05.
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B activation in mantle cell lymphoma B cells leads to induction of cell cycle arrest and apoptosis. J Immunol 2003;171:88–95.-2b in advanced melanoma outpatients: results from an Italian multicenter phase III randomized clinical trial. J Clin Oncol 2002;20:1600–7.B mutations or activation of the CD30, CD40, and RANK receptors. Clin Cancer Res 2004;10:3207–15.B:NF-B complexes in the resistance of cancer cells to Taxol-induced apoptosis. Oncogene 2002;21:6510–9.[CrossRef][Medline]
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D. L. Hodge, J. Yang, M. D. Buschman, P. M. Schaughency, H. Dang, W. Bere, Y. Yang, R. Savan, J. J. Subleski, X.-M. Yin, et al. Interleukin-15 Enhances Proteasomal Degradation of Bid in Normal Lymphocytes: Implications for Large Granular Lymphocyte Leukemias Cancer Res., May 1, 2009; 69(9): 3986 - 3994. [Abstract] [Full Text] [PDF]
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G. B. Lesinski, E. T. Raig, K. Guenterberg, L. Brown, M. R. Go, N. N. Shah, A. Lewis, M. Quimper, E. Hade, G. Young, et al. IFN-{alpha} and Bortezomib Overcome Bcl-2 and Mcl-1 Overexpression in Melanoma Cells by Stimulating the Extrinsic Pathway of Apoptosis Cancer Res., October 15, 2008; 68(20): 8351 - 8360. [Abstract] [Full Text] [PDF]
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S. T. Nawrocki, J. S. Carew, K. H. Maclean, J. F. Courage, P. Huang, J. A. Houghton, J. L. Cleveland, F. J. Giles, and D. J. McConkey Myc regulates aggresome formation, the induction of Noxa, and apoptosis in response to the combination of bortezomib and SAHA Blood, October 1, 2008; 112(7): 2917 - 2926. [Abstract] [Full Text] [PDF]
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N. Doudican, A. Rodriguez, I. Osman, and S. J. Orlow Mebendazole Induces Apoptosis via Bcl-2 Inactivation in Chemoresistant Melanoma Cells Mol. Cancer Res., August 1, 2008; 6(8): 1308 - 1315. [Abstract] [Full Text] [PDF]
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P. E. Lovat, M. Corazzari, J. L. Armstrong, S. Martin, V. Pagliarini, D. Hill, A. M. Brown, M. Piacentini, M. A. Birch-Machin, and C. P.F. Redfern Increasing Melanoma Cell Death Using Inhibitors of Protein Disulfide Isomerases to Abrogate Survival Responses to Endoplasmic Reticulum Stress Cancer Res., July 1, 2008; 68(13): 5363 - 5369. [Abstract] [Full Text] [PDF]
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 A. Sekulic, P. Haluska Jr, A. J. Miller, J. G. De Lamo, S. Ejadi, J. S. Pulido, D. R. Salomao, E. C. Thorland, R. G. Vile, D. L. Swanson, et al. Malignant Melanoma in the 21st Century: The Emerging Molecular Landscape Mayo Clin. Proc., July 1, 2008; 83(7): 825 - 846. [Abstract] [Full Text] [PDF]
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C. Tse, A. R. Shoemaker, J. Adickes, M. G. Anderson, J. Chen, S. Jin, E. F. Johnson, K. C. Marsh, M. J. Mitten, P. Nimmer, et al. ABT-263: A Potent and Orally Bioavailable Bcl-2 Family Inhibitor Cancer Res., May 1, 2008; 68(9): 3421 - 3428. [Abstract] [Full Text] [PDF]
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M. A. Nikiforov, M. Riblett, W.-H. Tang, V. Gratchouck, D. Zhuang, Y. Fernandez, M. Verhaegen, S. Varambally, A. M. Chinnaiyan, A. J. Jakubowiak, et al. Tumor cell-selective regulation of NOXA by c-MYC in response to proteasome inhibition PNAS, December 4, 2007; 104(49): 19488 - 19493. [Abstract] [Full Text] [PDF]
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J. Voortman, E. F. Smit, R. Honeywell, B. C. Kuenen, G. J. Peters, H. van de Velde, and G. Giaccone A Parallel Dose-Escalation Study of Weekly and Twice-Weekly Bortezomib in Combination with Gemcitabine and Cisplatin in the First-Line Treatment of Patients with Advanced Solid Tumors Clin. Cancer Res., June 15, 2007; 13(12): 3642 - 3651. [Abstract] [Full Text] [PDF]
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J. Han, L. A. Goldstein, W. Hou, and H. Rabinowich Functional Linkage between NOXA and Bim in Mitochondrial Apoptotic Events J. Biol. Chem., June 1, 2007; 282(22): 16223 - 16231. [Abstract] [Full Text] [PDF]
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J. Voortman, A. Checinska, G. Giaccone, J. A. Rodriguez, and F. A.E. Kruyt Bortezomib, but not cisplatin, induces mitochondria-dependent apoptosis accompanied by up-regulation of noxa in the non-small cell lung cancer cell line NCI-H460 Mol. Cancer Ther., March 1, 2007; 6(3): 1046 - 1053. [Abstract] [Full Text] [PDF]
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S. Meister, U. Schubert, K. Neubert, K. Herrmann, R. Burger, M. Gramatzki, S. Hahn, S. Schreiber, S. Wilhelm, M. Herrmann, et al. Extensive Immunoglobulin Production Sensitizes Myeloma Cells for Proteasome Inhibition Cancer Res., February 15, 2007; 67(4): 1783 - 1792. [Abstract] [Full Text] [PDF]
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L. A. Smit, D. Y.H. Hallaert, R. Spijker, B. de Goeij, A. Jaspers, A. P. Kater, M. H.J. van Oers, C. J.M. van Noesel, and E. Eldering Differential Noxa/Mcl-1 balance in peripheral versus lymph node chronic lymphocytic leukemia cells correlates with survival capacity Blood, February 15, 2007; 109(4): 1660 - 1668. [Abstract] [Full Text] [PDF]
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M. Verhaegen, J. A. Bauer, C. Martin de la Vega, G. Wang, K. G. Wolter, J. C. Brenner, Z. Nikolovska-Coleska, A. Bengtson, R. Nair, J. T. Elder, et al. A Novel BH3 Mimetic Reveals a Mitogen-Activated Protein Kinase-Dependent Mechanism of Melanoma Cell Death Controlled by p53 and Reactive Oxygen Species Cancer Res., December 1, 2006; 66(23): 11348 - 11359. [Abstract] [Full Text] [PDF]
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J. Hur, D. W. Bell, K. L. Dean, K. R. Coser, P. C. Hilario, R. A. Okimoto, E. M. Tobey, S. L. Smith, K. J. Isselbacher, and T. Shioda Regulation of Expression of BIK Proapoptotic Protein in Human Breast Cancer Cells: p53-Dependent Induction of BIK mRNA by Fulvestrant and Proteasomal Degradation of BIK Protein. Cancer Res., October 15, 2006; 66(20): 10153 - 10161. [Abstract] [Full Text] [PDF]
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J.-Z. Qin, H. Xin, L. A. Sitailo, M. F. Denning, and B. J. Nickoloff Enhanced Killing of Melanoma Cells by Simultaneously Targeting Mcl-1 and NOXA Cancer Res., October 1, 2006; 66(19): 9636 - 9645. [Abstract] [Full Text] [PDF]
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 C. S. Mitsiades, D. McMillin, V. Kotoula, V. Poulaki, C. McMullan, J. Negri, G. Fanourakis, S. Tseleni-Balafouta, K. B. Ain, and N. Mitsiades Antitumor Effects of the Proteasome Inhibitor Bortezomib in Medullary and Anaplastic Thyroid Carcinoma Cells in Vitro J. Clin. Endocrinol. Metab., October 1, 2006; 91(10): 4013 - 4021. [Abstract] [Full Text] [PDF]
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E. Aleo, C. J. Henderson, A. Fontanini, B. Solazzo, and C. Brancolini Identification of new compounds that trigger apoptosome-independent caspase activation and apoptosis. Cancer Res., September 15, 2006; 66(18): 9235 - 9244. [Abstract] [Full Text] [PDF]
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X. Dolcet, D. Llobet, M. Encinas, J. Pallares, A. Cabero, J. A. Schoenenberger, J. X. Comella, and X. Matias-Guiu Proteasome Inhibitors Induce Death but Activate NF-{kappa}B on Endometrial Carcinoma Cell Lines and Primary Culture Explants J. Biol. Chem., August 4, 2006; 281(31): 22118 - 22130. [Abstract] [Full Text] [PDF]
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J. A. Sosman and I. Puzanov Molecular targets in melanoma from angiogenesis to apoptosis. Clin. Cancer Res., April 1, 2006; 12(7): 2376s - 2383s. [Abstract] [Full Text] [PDF]
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Y. Fernandez, T. P. Miller, C. Denoyelle, J. A. Esteban, W.-H. Tang, A. L. Bengston, and M. S. Soengas Chemical Blockage of the Proteasome Inhibitory Function of Bortezomib: IMPACT ON TUMOR CELL DEATH J. Biol. Chem., January 13, 2006; 281(2): 1107 - 1118. [Abstract] [Full Text] [PDF]
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 G. C. Shore and J. Viallet Modulating the Bcl-2 Family of Apoptosis Suppressors for Potential Therapeutic Benefit in Cancer Hematology, January 1, 2005; 2005(1): 226 - 230. [Abstract] [Full Text] [PDF]
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