Previously, we have shown that bortezomib overcame TRAIL resistance in hepatocellular carcinoma (HCC) cells via the inhibition of Akt. Here, we report that bortezomib sensitizes these TRAIL-resistant cells, including Huh-7, Hep3B, and Sk-Hep1, to CS-1008, a humanized agonistic antihuman death receptor 5 antibody. Cancerous inhibitor of protein phosphatase 2A (CIP2A) mediated the sensitizing effect of bortezomib to CS-1008 through inhibiting protein phosphatase 2A (PP2A) activity. Combination treatment of bortezomib and CS-1008 downregulated CIP2A in a concentration- and time-dependent manner, and increased PP2A activity in HCC cells. Importantly, ectopic expression of CIP2A decreased Akt-related PP2A activity, indicating that CIP2A negatively regulates Akt-related PP2A activity in HCC cells. Moreover, silencing CIP2A by short interfering RNA enhanced CS-1008–induced apoptosis in HCC cells and ectopic expression of CIP2A in HCC cells abolished CS-1008–induced apoptosis, indicating that CIP2A plays an important role in the sensitizing effect of bortezomib to CS-1008. Finally, our in vivo data showed that CS-1008 and bortezomib combination treatment decreased tumor growth significantly. In conclusion, bortezomib sensitized HCC cells to CS-1008 through the inhibition of CIP2A. Mol Cancer Ther; 10(5); 892–901. ©2011 AACR.

Hepatocellular carcinoma (HCC) is the fifth most common solid tumor worldwide and remains a difficult malignancy to treat (1). Major obstacles hindering effective treatment for HCC include high recurrence after curative resection, underlying cirrhotic liver, and, importantly, the frequent resistance of advanced HCC to conventional chemotherapy and radiotherapy (2). The fact that most patients with advanced HCC do not respond well to current chemotherapeutic agents highlights the need for the development of novel targeted therapy for HCC (3). And, the promising trial result shown by the multikinase inhibitor sorafenib in patients with advanced HCC showed the potential of molecular-targeted therapy for advanced HCC (4).

Targeting apoptotic pathway is the foundation of many anticancer strategies. Among the possibilities, the pathway involving TNF-related apoptosis inducing ligand (TRAIL) is currently the most promising, as preclinical models suggest that apoptosis of tumor cells is achievable in vivo without lethal toxicities (5). TRAIL is a type II transmembrane protein that belongs to the TNF superfamily and functions through binding to death receptors (DR; ref. 6). Currently, 5 DRs that can be subdivided into 2 distinctive functional groups are known to bind to TRAIL (7, 8). DR4 (TRAIL-R1) and DR5 (TRAIL-R2) contain an effective cytoplasmic death domain that forms death-inducing signaling complex (DISC) on ligand binding and transduce proapoptotic signals (7, 8). The binding of TRAIL to DR4 or DR5 results in trimerization of the death domain-containing receptor, leading to the formation of a multiprotein complex designated the DISC, which involves participation of an adaptor molecule, Fas-associated protein with death domain (FADD) and activation of the initiator caspase-8.

CS-1008, a novel humanized agonistic antihuman DR5 antibody, was modified from a murine antihuman DR5 mAb, TRA-8 (9). CS-1008 has shown selective cytotoxicity toward tumor cells expressing DR5, such as colorectal adenocarcinoma cells, non–small-cell lung cancer cells, pancreatic carcinoma cells, and renal cell adenocarcinoma and has shown enhanced antitumor activity when in combination with gemcitabine or docetaxel (9). In a phase I trial, CS-1008 exerted no dose limiting toxicity at doses of up to 8 mg/kg weekly (10). CS-1008 is currently undergoing phase II trials in combination with sorafenib for the treatment of advanced HCC (NCI clinical trial: NCT01033240).

Despite reports of selective TRAIL-induced apoptosis in several types of tumor cells, many cancer cells are resistant to apoptosis induction by TRAIL and more and more evidence suggest that TRAIL alone may not be sufficient to efficiently induce apoptosis in many types of cancers, including HCC (11–14). Therefore, it is important both to understand the mechanisms underlying resistance and improve the potency of TRAIL-based therapeutic approaches, if TRAIL is going to be successfully used for cancer therapy. Proteasome inhibitors represent a highly promising class of anticancer agents to overcome TRAIL resistance or resensitize tumors to the apoptotic effect of TRAIL.

Bortezomib is the first proteasome inhibitor to be approved clinically for multiple myeloma and mantel cell lymphoma (15, 16). Bortezomib showed excellent antitumor activity against these 2 hematologic malignancies through blocking proteasome degradation of IκB, an inhibitor of NF-κB (16). The fact that multiple cellular targets are affected by bortezomib suggests its potential advantage in enhancing antitumor activities in combination treatment with TRAIL in HCC. Indeed, in our previous study, exploring the antitumor activity of bortezomib against HCC cells (17), we showed that downregulation of p-Akt determines the sensitivity of bortezomib, and that bortezomib-induced apoptosis may not be associated with proteasome inhibition in HCC cells. Moreover, we confirmed that bortezomib sensitized HCC cells to TRAIL through inhibition of p-Akt (14). Importantly, we found that protein phosphatase 2A (PP2A), a phosphatase which downregulates p-Akt, may mediate the effect of bortezomib on TRAIL sensitization (14). The mechanism by which bortezomib upregulates PP2A and, in turn, downregulates p-Akt was further delineated in our recent study on the synergistic interaction between bortezomib and sorafenib in HCC cells (18).

PP2A is a complex of serine/threonine protein phosphatase with broad substrate specificity and diverse cellular functions and consists of a dimeric core enzyme comprising of structural A and catalytic C subunits (the AC core enzyme), and heterogeneous regulatory B subunits (19, 20). It is known that there are several cellular inhibitors of PP2A, including SET (21) and cancerous inhibitor of PP2A (CIP2A; ref. 22). Notably, CIP2A (KIAA1524, P90), first cloned from patients with HCC (23), has been shown to promote anchorage-independent cell growth and in vivo tumor formation by inhibiting PP2A activity toward c-myc (22). Moreover, CIP2A is overexpressed in HCC and several other human malignancies (22, 24, 25) and is associated with clinical aggressiveness in human breast cancer (24), suggesting its role as an oncoprotein.

In this study, we found that bortezomib sensitizes TRAIL-resistant HCC cells to CS-1008 through the mediator CIP2A. We showed that CIP2A, through inhibition of PP2A-dependent p-Akt activity, mediates the sensitizing effect of bortezomib to CS-1008. The combination of bortezomib and CS-1008 downregulated CIP2A and, in turn, increased Akt-related PP2A activity in these HCC cells.

Reagents and antibodies

Bortezomib (Velcade) and CS-1008 were kindly provided by Millennium Pharmaceuticals and Daiichi Sankyo Pharmaceuticals, respectively. For in vitro studies, bortezomib or CS-1008 at various concentrations was dissolved in dimethyl sulfoxide (DMSO) and then added to cells in Dulbecco's Modified Eagle's Medium (DMEM) containing 5% FBS. The final DMSO concentration was 0.1% after addition to the medium. Antibodies for immunoblotting such as anti-Akt1, anti-PARP, and anti–PP2A-C, were purchased from Santa Cruz Biotechnology. Other antibodies including anti–caspase-3, anti-CIP2A, and anti–p-Akt (Ser473) were from Cell Signaling.

Cell culture and Western blot analysis

The Sk-Hep1 and Hep3B cell lines were obtained from the American Type Culture Collection. The Huh-7 HCC cell line was obtained from the Health Science Research Resources Bank (Osaka, Japan; JCRB0403). All cells obtained from the American Type Culture Collection or the Health Science Research Resources Bank were immediately expanded and frozen down such that all cell lines could be restarted every 3 months from a frozen vial of the same batch of cells. No further authentication was done in our lab. Cells were maintained in DMEM supplemented with 10% FBS, 100 units/mL penicillin G, 100 μg/mL streptomycin sulfate, and 25 μg/mL amphotericin B in a 37°C humidified incubator and an atmosphere of 5% CO2 in air. Lysates of HCC cells treated with drugs at the indicated concentrations for various periods of time were prepared for immunoblotting of caspase-3, PARP, p-Akt, and Akt. Western blot analysis was done as previously reported (17).

Apoptosis analysis

The following 3 methods were used to assess drug-induced apoptotic cell death: detection of DNA fragmentation with the Cell Death Detection ELISA kit (Roche Diagnostics), Western blot analysis of caspase activation and PARP cleavage, and measurement of apoptotic cells by flow cytometry (sub-G1). ELISA was conducted according to the manufacturer's instructions (8).

Gene knockdown using short interfering RNA

Smartpool short-interfering RNA (siRNA) reagents, including a control (D-001810-10), and CIP2A (L-014135-01) were all purchased from Dharmacon and used according to the procedure described previously (17). Briefly, cells were transfected with siRNA (final concentration, 100 nmol/L) in 6-well plates using the Dharma-FECT4 transfection reagent (Dharmacon), according to the manufacturer's instructions. After 48 hours, the medium was replaced and the HCC cells were incubated with bortezomib, harvested, and separated for Western blot analysis and for apoptosis analysis by flow cytometry as described previously (17).

Sk-Hep1 with constitutive active CIP2A

CIP2A cDNA (KIAA1524) was purchased from Origene (RC219918). Briefly, following transfection, cells were incubated in the presence of G418 (0.78 mg/mL). After 8 weeks of selection, surviving colonies, that is, those arising from stably transfected cells, were selected and individually amplified. Sk-Hep1 cells, with stable expression of CIP2A-myc, were then treated with bortezomib, harvested, and processed for Western blot analysis as described previously.

Coimmunoprecipitation assay

Cells were harvested and lysed on ice for 30 minutes in lysis buffer (50 mmol/L Tris-HCl, pH 7.4, 100 mmol/L NaCl, 0.5% Nonidet P-40, 50 mmol/L NaF, 1 mmol/L Na3VO4, 5 mmol/L sodium pyrophosphate, and a protease inhibitor tablet). The cell lysates were centrifuged at 14,000 × g for 15 minutes, and the supernatants were recovered. Supernatants containing equal amounts of proteins were incubated with 2.5 mg of primary antibodies overnight at 4°C. The immunoprecipitates were harvested using protein G PLUS-agarose beads (Santa Cruz Biotechnology) that were washed once with regular washing buffer (50 mmol/L Tris-HCl, 100 mmol/L NaCl, 1 mmol/L EDTA, and 0.5% Nonidet P-40), twice with high salt washing buffer (50 mmol/L Tris-HCl, 500 mmol/L NaCl, 1 mmol/L EDTA, and 0.5% Nonidet P-40),and once again with regular washing buffer. Immunoprecipitates were then eluted by boiling the beads for 5 minutes in SDS-PAGE sample buffer and characterized by Western blotting with appropriate antibodies.

PP2A phosphatase activity

The protein phosphatase activity in total cellular lysate was determined by measuring the generation of free phosphate from threonine phosphopeptide using the malachite green–phosphate complex assay as described by the manufacturer (Upstate Biotechnology). Cell lysates were prepared in a low-detergent lysis buffer (1% Nonidet P-40, 10 mmol/L HEPES, 150 mmol/L NaCl, 10% glycerol, 1 mmol/L phenylmethylsulfonyl fluoride, 5 mmol/L benzamidine, and 10 g/mL leupeptin). The phosphatase assay was done in a PP2A-specific reaction buffer (Upstate) containing 750 μmol/L phosphopeptide substrate. After 10 minutes of incubation at 30°C, the malachite dye was added, and free phosphate was measured by optical density at 650 nm. To avoid variability due to differences in the amounts of immunoprecipitated protein between samples, the phosphatase activities were normalized to the amount of PP2A immunoprecipitated, as detected and quantified by immunoblot analysis for each treatment group.

Immunofluorescent staining

Sk-Hep1 cells (wild-type or CIP2A-myc) were seeded on sterilized slides in a 10-cm dish overnight and then treated with DMSO or okadaic acid (wild-type) or forskolin (CIP2A-myc) for 24 hours. The cells were washed with PBS, fixed with 4% formaldehyde for 10 minutes at 37°C, and then washed with PBS twice. Cells were treated with 0.1% Triton X-100 and then blocked with 0.5% bovine serum albumin (BSA) in PBS at 37°C for 1 hour. Then cells were treated with anti-CIP2A or anti–p-Akt antibody (1:50 in PBS containing 0.5% BSA) at 4°C overnight. Fluorescein isothiocyanate–conjugated goat anti-mouse IgG (CIP2A; 1:1,000) or anti-rabbit IgG (p-Akt; 1:1,000) was applied for 1 hour at room temperature. The cells were observed under a fluorescence microscope (Leica DM2500).

Xenograft tumor growth

Male NCr athymic nude mice (5–7 weeks of age) were obtained from the National Laboratory Animal Center (Taipei, Taiwan). The mice were housed in groups and maintained under standard laboratory conditions on a 12-hour light-dark cycle. They were given access to sterilized food and water ad libitum. All experimental procedures using these mice were conducted in accordance with protocols approved by the Institutional Laboratory Animal Care and Use Committee of National Taiwan University. Each mouse was inoculated subcutaneously in the dorsal flank, with 1 × 106 HCC cells suspended in 0.1 mL of serum-free medium containing 50% Matrigel (BD Biosciences). When tumors reached 150 to 200 mm3, mice were randomized divided into 4 groups (n = 8) and received an intraperitoneal injection of bortezomib (0.5 mg/kg body weight) twice weekly and/or intravenous injection of CS-1008 200 μg 3 times a week. Controls received vehicle consisting of 0.5% methylcellulose and 0.1% polysorbate 80 in sterile water. Data are representative of 3 independent experiments.

Statistical analysis

Tumor growth data points are reported as mean tumor volume ± SE. Comparisons of mean values were done using the independent samples t test in SPSS for Windows 11.5 software (SPSS Inc.).

Bortezomib enhances CS-1008–induced apoptosis in HCC cells

To investigate the antitumor effect of CS-1008 on HCC cells, we first examined the apoptotic effect of CS-1008 in a panel of 3 human HCC cell lines, Huh-7, Sk-Hep1, and Hep3B at the clinical relevant concentrations. Apoptotic cells (sub-G1) were determined by flow cytometry after 24 hours treatment. As shown in Fig. 1, HCC cells were quite resistant to CS-1008 and treatment with CS-1008 alone could not efficiently induce apoptosis in these HCC cells, even up to a concentration of 800 ng/mL. However, combining bortezomib at 50 nmol/L with CS-1008 overcame the resistance and sensitized cells to apoptosis significantly in a concentration-dependent manner, starting from CS-1008 at a concentration of 200 ng/mL. We then examined the effect of a fixed concentration of CS-1008 on escalating concentrations of bortezomib and found that CS-1008 at a dose of 200 ng/mL increased bortezomib-induced apoptosis in a concentration-dependent manner (Fig. 1B).

Figure 1

Bortezomib enhances CS-1008–induced apoptosis in HCC cells. A, concentration-escalation effects of CS-1008 with 50 nmol/L bortezomib on apoptosis in 3 TRAIL-resistant HCC cells. B, concentration-escalation effects of bortezomib with 200 ng/mL CS-1008. HCC cells were exposed to bortezomib and/or CS-1008 at the indicated concentrations in DMEM, with 5% FBS in 6-well plates for 24 hours, and apoptotic cells were assessed by flow cytometry. Points, mean; bars, SD (n = 3). C, effects of bortezomib on DNA fragmentation in HCC. Cells were exposed to CS-1008 (200 ng/mL) and/or bortezomib at the indicated concentrations (nmol/L) in DMEM with 5% FBS for 24 hours. Cell lysates were prepared and analyzed for cell death by ELISA. Data are representative of 3 independent experiments. D, the chemical structure of bortezomib.

Figure 1

Bortezomib enhances CS-1008–induced apoptosis in HCC cells. A, concentration-escalation effects of CS-1008 with 50 nmol/L bortezomib on apoptosis in 3 TRAIL-resistant HCC cells. B, concentration-escalation effects of bortezomib with 200 ng/mL CS-1008. HCC cells were exposed to bortezomib and/or CS-1008 at the indicated concentrations in DMEM, with 5% FBS in 6-well plates for 24 hours, and apoptotic cells were assessed by flow cytometry. Points, mean; bars, SD (n = 3). C, effects of bortezomib on DNA fragmentation in HCC. Cells were exposed to CS-1008 (200 ng/mL) and/or bortezomib at the indicated concentrations (nmol/L) in DMEM with 5% FBS for 24 hours. Cell lysates were prepared and analyzed for cell death by ELISA. Data are representative of 3 independent experiments. D, the chemical structure of bortezomib.

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We further examined these apoptotic effects using an ELISA kit which detects DNA fragmentation. Our data showed that combination of CS-1008 and bortezomib increased DNA fragmentation in a dose- and time-dependent manner (Fig. 1C). These results indicate that a combination of CS-1008 and bortezomib overcame the resistance of HCC cells to the TRAIL receptor antibody, CS-1008.

Downregulation of CIP2A is associated with bortezomib and CS-1008 combination treatment in HCC cells

Our previous data suggested that the sensitizing effect of bortezomib on TRAIL resistance of HCC cells was mediated by the PP2A-Akt signaling pathway (14). Because CIP2A inhibits PP2A, we further hypothesized that inhibition of CIP2A may be associated with the sensitizing effect of bortezomib on CS-1008–induced apoptosis in HCC cells. As shown in Fig. 2A, combined treatment of bortezomib and CS-1008 reduced protein levels of CIP2A, in correlation with downregulation of p-Akt expression and induced apoptosis in all tested HCC cells. Evidence for the association between CIP2A inhibition and apoptosis induction was further strengthened by the time-dependent PARP cleavage in the combined bortezomib- and CS-1008–treated HCC cells (Fig. 2B). These results suggest that inhibition of CIP2A plays a key role in the mediating effects of the drug combination.

Figure 2

Downregulation of CIP2A is associated with sensitizing effects of bortezomib in HCC cells. A, effects of bortezomib on protein levels of CIP2A, p-Akt, Akt, and Bcl-2 family in HCC cells. HCC cells were treated with bortezomib (100 nmol/L) and/or CS-1008 (200 ng/mL) for 24 hours and cell lysates were prepared for Western blot. Data are representative of 3 independent experiments. B, time-dependent analysis of CIP2A level and apoptotic death with bortezomib and CS-1008 combination treatment. Sk-Hep1 cells were exposed to bortezomib and/or CS-1008 for the indicated period of time. Cell lysates were prepared and assayed for CIP2A, p-Akt, Akt, and PARP. CF, cleaved form (activated form).

Figure 2

Downregulation of CIP2A is associated with sensitizing effects of bortezomib in HCC cells. A, effects of bortezomib on protein levels of CIP2A, p-Akt, Akt, and Bcl-2 family in HCC cells. HCC cells were treated with bortezomib (100 nmol/L) and/or CS-1008 (200 ng/mL) for 24 hours and cell lysates were prepared for Western blot. Data are representative of 3 independent experiments. B, time-dependent analysis of CIP2A level and apoptotic death with bortezomib and CS-1008 combination treatment. Sk-Hep1 cells were exposed to bortezomib and/or CS-1008 for the indicated period of time. Cell lysates were prepared and assayed for CIP2A, p-Akt, Akt, and PARP. CF, cleaved form (activated form).

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Target validation of CIP2A

Two different approaches were used to validate the role of CIP2A in the sensitizing effect of bortezomib on CS-1008–induced apoptosis in HCC cells. First, we knocked down protein expression of CIP2A by using siRNA. Sk-Hep1 cells were transfected with either scrambled siRNA as a control or CIP2A siRNA for 48 hours and were then exposed to DMSO or CS-1008 (200 ng/mL) for another 24 hours. As shown in Fig. 3A, depletion of CIP2A by siRNA significantly reduced the resistance of CS-1008 in Sk-Hep1 cells in tandem with downregulation of p-Akt (Ser473) and enhanced apoptotic cell death. Notably, knockdown of CIP2A alone or in combination with CS-1008 showed similar effects on downregulation of p-Akt, suggesting that adding CS-1008 does not enhance the effect of CIP2A on p-Akt. Next, we generated Sk-Hep1 (CIP2A-myc) cells with stably expressed CIP2A to investigate the sensitizing effect of bortezomib. Ectopic expression of CIP2A significantly abolished the sensitizing effect of bortezomib on CS-1008–induced apoptosis in comparison with wild-type Sk-Hep1 cells that underwent the same treatment (P < 0.05; Fig. 3B). Together, these results validated the importance of CIP2A inhibition in mediating the effect of bortezomib on CS-1008 sensitivity to HCC cells.

Figure 3

In vitro target validation of CIP2A. A, downregulation of CIP2A by siRNA overcomes the resistance to CS-1008 in Sk-Hep1 cells. Bottom, protein levels of CIP2A. Top, analysis of apoptotic cells. Cells were transfected with either control siRNA or CIP2A siRNA for 48 hours and then exposed to CS-1008 (200 ng/mL) for 24 hours. For analysis of apoptotic cells (sub-G1), cells were analyzed by flow cytometry. Columns, mean; bars, SD (n = 3). B, protective effects of ectopic expression of CIP2A on apoptosis induced by the combination of CS-1008 and bortezomib in Sk-Hep1 cells. Right, protein levels of Akt in both wild-type and Huh7-Akt cells. WT, wild-type. Left, analysis of apoptotic cells. Columns, mean; bars, SD (n = 3). Sk-Hep1 cells were transfected with CIP2A with myc-tag and were selected for 8 weeks by G-418. Analysis of apoptotic cells was done by flow cytometry after cells were exposed to the combination of bortezomib 100 nmol/L and CS-1008 200 ng/mL for 24 hours.

Figure 3

In vitro target validation of CIP2A. A, downregulation of CIP2A by siRNA overcomes the resistance to CS-1008 in Sk-Hep1 cells. Bottom, protein levels of CIP2A. Top, analysis of apoptotic cells. Cells were transfected with either control siRNA or CIP2A siRNA for 48 hours and then exposed to CS-1008 (200 ng/mL) for 24 hours. For analysis of apoptotic cells (sub-G1), cells were analyzed by flow cytometry. Columns, mean; bars, SD (n = 3). B, protective effects of ectopic expression of CIP2A on apoptosis induced by the combination of CS-1008 and bortezomib in Sk-Hep1 cells. Right, protein levels of Akt in both wild-type and Huh7-Akt cells. WT, wild-type. Left, analysis of apoptotic cells. Columns, mean; bars, SD (n = 3). Sk-Hep1 cells were transfected with CIP2A with myc-tag and were selected for 8 weeks by G-418. Analysis of apoptotic cells was done by flow cytometry after cells were exposed to the combination of bortezomib 100 nmol/L and CS-1008 200 ng/mL for 24 hours.

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CIP2A inhibits Akt-associated PP2A activity

Our previous work showed that bortezomib increased PP2A activity in HCC cells (14, 18). We, thus, next examined how bortezomib and CS-1008 combination treatment affected PP2A activity. As shown in Fig. 4A, bortezomib plus CS-1008 significantly upregulated PP2A activity in Sk-Hep1 and Huh-7 cells. Moreover, we found that treatment with either of the drugs alone or in combination did not significantly affect the protein level of PP2A complex including subunit A, B56γ, and C in Sk-Hep1 and Hep3B cells, except the drugs combination decreased PP2A-B55y in Huh-7 cells (Fig. 4B). These data suggested that the alterations of PP2A complex may not play a major role in mediating the sensitizing effect of bortezomib on CS-1008–treated HCC cells. Notably, CS-1008 and bortezomib combination treatment, compared with either drug alone, did not alter protein–protein interactions between Akt and PP2A significantly, suggesting that CIP2A does not affect their binding affinity directly (Fig. 4C). In addition, previous study has indicated that CIP2A inhibits c-myc–associated PP2A activity thereby stabilizing c-myc (22). We, therefore, similarly examined Akt-related PP2A activity by coimmunoprecipitation of Akt in Sk-Hep1 cells. Our data indicated that ectopic expression of CIP2A reduced PP2A activity on Akt, suggesting that CIP2A plays a role in regulating Akt-related PP2A activity (Fig. 4D, left). In addition, the data of immunofluoresence staining also showed that the CIP2A-myc cells had higher p-Akt than the wild-type cells (Fig. 4D, right). The treatment of okadaic acid (a PP2A inhibitor) in wild-type cells enhanced the expression of p-Akt, whereas forskolin (a PP2A agonist) reduced the expression of p-Akt in CIP2A-myc cells (Fig. 4D, right). These data indicate that CIP2A reduces the PP2A activity on Akt in HCC cells.

Figure 4

CIP2A inhibits Akt-associated PP2A activity. A, analysis of PP2A activity in HCC cells. Columns, mean; bars, SD (n = 3). Cells (Sk-Hep1 or Huh-7) were treated with DMSO or bortezomib (Btz; 100 nmol/L) and CS-1008 (200 ng/mL) for 24 hours. Cell lysates were prepared for detecting PP2A activity. B, effects of bortezomib and/or CS-1008 on protein levels of PP2A complex. Cells were exposed to bortezomib (100 nmol/L) and/or CS-1008 (200 ng/mL) for 24 hours. C, effects of bortezomib on the dynamic PP2A–Akt interactions in Sk-Hep1 cells. Cells were treated with bortezomib (100 nmol/L) and/or CS-1008 (200 ng/mL) for 24 hours, and cell lysates were immunoprecipitated with anti–PP2A-C antibodies. The immunoprecipitates were probed with Akt1 and PP2A-C by Western blot. D, left, overexpression of CIP2A decreased Akt-associated PP2A activity. Cell lysates from Sk-Hep1 cells (wild-type or CIP2A-myc) were immunoprecipitated with anti-Akt1 antibodies. Cell lysates were prepared for detecting PP2A activity. Columns, mean; bars, SD (n = 3). Right, CIP2A and p-Akt staining. Top, Sk-Hep1 wild-type cells; bottom, CIP2A overexpression cells. OA, okadaic acid (a PP2A inhibitor). Forskolin, a PP2A agonist. Cells were treated with DMSO or OA (wild-type) or forskolin (CIP2A-myc) for 24 hours then stained with CIP2A (red) or p-Akt (green) and examined under a fluorescence microscope.

Figure 4

CIP2A inhibits Akt-associated PP2A activity. A, analysis of PP2A activity in HCC cells. Columns, mean; bars, SD (n = 3). Cells (Sk-Hep1 or Huh-7) were treated with DMSO or bortezomib (Btz; 100 nmol/L) and CS-1008 (200 ng/mL) for 24 hours. Cell lysates were prepared for detecting PP2A activity. B, effects of bortezomib and/or CS-1008 on protein levels of PP2A complex. Cells were exposed to bortezomib (100 nmol/L) and/or CS-1008 (200 ng/mL) for 24 hours. C, effects of bortezomib on the dynamic PP2A–Akt interactions in Sk-Hep1 cells. Cells were treated with bortezomib (100 nmol/L) and/or CS-1008 (200 ng/mL) for 24 hours, and cell lysates were immunoprecipitated with anti–PP2A-C antibodies. The immunoprecipitates were probed with Akt1 and PP2A-C by Western blot. D, left, overexpression of CIP2A decreased Akt-associated PP2A activity. Cell lysates from Sk-Hep1 cells (wild-type or CIP2A-myc) were immunoprecipitated with anti-Akt1 antibodies. Cell lysates were prepared for detecting PP2A activity. Columns, mean; bars, SD (n = 3). Right, CIP2A and p-Akt staining. Top, Sk-Hep1 wild-type cells; bottom, CIP2A overexpression cells. OA, okadaic acid (a PP2A inhibitor). Forskolin, a PP2A agonist. Cells were treated with DMSO or OA (wild-type) or forskolin (CIP2A-myc) for 24 hours then stained with CIP2A (red) or p-Akt (green) and examined under a fluorescence microscope.

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Effect of bortezomib on HCC xenograft tumor

To confirm whether the sensitizing effect of bortezomib on CS-1008 has potentially relevant clinical implications, we assessed the in vivo effect of bortezomib and CS-1008 on Huh-7 xenograft tumors. Tumor-bearing mice were treated with vehicle or bortezomib at 0.5 mg/kg i.p. twice a week and/or CS-1008 200 μg 3 times a week for the duration of the in vivo experiment. Throughout the course of treatment, all mice tolerated the treatments well without observable signs of toxicity and had stable body weights. No gross pathologic abnormalities were noted at necropsy. As shown in Fig. 5A, tumor growth was significantly inhibited by treatment with bortezomib plus CS-1008 for 2 weeks (vs. control, P < 0.05), and tumor size in the treatment group was only half that in the control group at the end of the study. Treatment with CS-1008 alone had no significant effect on Huh-7 tumor growth (vs. control, P > 0.05) and bortezomib alone showed modest effect. Moreover, as shown in Fig. 5B, CIP2A protein levels decreased significantly in Huh-7 tumors treated with bortezomib in combination with CS-1008. Finally, an analysis of the effect of bortezomib on PP2A activity (Fig. 5C) showed a significant increase in PP2A phosphatase activity (P < 0.05) in Huh-7 tumors treated with bortezomib alone and bortezomib plus CS-1008. However, the treatment of CS-1008 alone did not show significant effects on CIP2A and the PP2A activity (vs. control, P > 0.05), which is consistent with previous in vitro findings (Fig. 2A and Fig. 3A). These data indicate mediation of the effect via PP2A-dependent Akt inactivation in vivo. Further clinical investigation is warranted.

Figure 5

In vivo effect of bortezomib on Huh-7 xeonograft nude mice. A, effects of bortezomib and/or CS-1008 on tumor growth. Points, mean (n = 6); bars, SE. Each mouse was inoculated subcutaneously in the dorsal flank with 1 million Huh-7 cells suspended in Matrigel. When the tumor reached a volume of 200 mm3, mice received treatments of vehicle or 0.5 mg/kg bortezomib i.p. twice weekly and/or CS-1008 200 μg i.v. 3 times a week for the duration of the study. B, Western blot analysis of CIP2A in Huh-7 and PLC5 tumors. C, analysis of PP2A activity in tumors. Values are means ± SD (n = 3). The figure is representative of 3 independent experiments.

Figure 5

In vivo effect of bortezomib on Huh-7 xeonograft nude mice. A, effects of bortezomib and/or CS-1008 on tumor growth. Points, mean (n = 6); bars, SE. Each mouse was inoculated subcutaneously in the dorsal flank with 1 million Huh-7 cells suspended in Matrigel. When the tumor reached a volume of 200 mm3, mice received treatments of vehicle or 0.5 mg/kg bortezomib i.p. twice weekly and/or CS-1008 200 μg i.v. 3 times a week for the duration of the study. B, Western blot analysis of CIP2A in Huh-7 and PLC5 tumors. C, analysis of PP2A activity in tumors. Values are means ± SD (n = 3). The figure is representative of 3 independent experiments.

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In this study, we confirmed that CS-1008, a novel anti-human DR5 antibody with promising TRAIL-like anticancer activity, is not sufficient to kill HCC cells as a monotherapy. However, we found that bortezomib effectively sensitizes HCC cells to this novel anti-DR5 antibody. Importantly, we delineated the mechanism by which bortezomib sensitizes these resistant cells to CS-1008. Our results have several important implications. First, CIP2A may serve as a potential drug target in HCC. We identified CIP2A inhibition as a major determinant of sensitizing effects of bortezomib which is dissociated from proteasome inhibition. This suggests that other novel agents that are capable of downregulating CIP2A protein expression may also be good sensitizers of TRAIL-resistant cells. Indeed, knockdown of CIP2A by siRNA was able to restore CS-1008–induced apoptosis in resistant Sk-Hep1 cells (Fig. 3A). Moreover, Sk-Hep1 cells with ectopic overexpression of CIP2A were more resistant to the effect of bortezomib and CS-1008 combination treatment (Fig. 3B). As an oncoprotein, CIP2A may promote cancer cell aggressiveness, thereby facilitating resistance of cancer cells to apoptotic-inducing agents. Currently, there are no known specific CIP2A inhibitors and whether CIP2A is suitable as predictive markers for drug therapy remain to be determined. Future preclinical research exploring agents that inhibit CIP2A and clinicopathologic studies correlating CIP2A expression and drug sensitivity in HCC patients are warranted to further consolidate CIP2A as a good drug target. Notably, the data from a phase I study in human indicate that CS-1008 is well tolerated and the Cmin of CS-1008 is 14 μg/mL (1 mg/kg/wk; ref. 26).

Various mechanisms contribute to TRAIL resistance in cancers (12). Resistance to TRAIL can occur at any step in the apoptosis signaling cascade. For example, receptor level mutations or overexpression of DR4 or DR5 can lead to resistance (12). Defects in members participating in DISC assembly, such as FADD, caspase-8, and cellular FADD-like interleukin-1b–converting enzyme inhibitory protein can also result in resistance to TRAIL (27, 28). Furthermore, overexpression of antiapoptotic Bcl-2 family proteins (Bcl-2, Bcl-XL, Mcl-1, etc; refs. 29, 30), loss of proapoptotic protein function (Bax or Bak), and an inability to activate mitochondria during apoptosis, such as reduced release of mitochondria-derived activator of caspases (SMAC/DIABLO), have been shown to cause TRAIL resistance in mitochondria-dependent type II cancer cells (12, 31). Finally, aberrantly activated antiapoptotic pathways in various tumor cells, such as phosphoinositol-3-kinase (PI3K)/Akt signaling, mitogen-activated protein kinases pathway and NF-κB may contribute to development of TRAIL resistance (12, 32, 33). Interestingly, we discovered that through CIP2A-PP2A-p-Akt regulatory mechanism, bortezomib overcomes CS-1008 resistance, supporting the hypothesis that constitutively active Akt signaling confers resistance of HCC cells to TRAIL and anti-DR5 CS-1008.

Previous literature, including our own work, has shown that bortezomib and other investigational proteasome inhibitors are capable of sensitizing cancer cells to TRAIL-induced apoptosis (14, 34–38). However, the molecular mechanisms underlying the sensitizing effect of bortezomib seem to be complicated and may be specific to cancer types. In certain cancer types, sensitization to TRAIL resistance has been reported to be through proteasome inhibition, including upregulation of TRAIL receptors (34, 38), activation of both extrinsic and intrinsic apoptosis pathways (39, 40), and probably, many other multiple molecular machineries. For example, Hetschko and colleagues (34) showed that the proteasome inhibitors MG132, via enhancement of transcription and surface expression of DR5, potentiates TRAIL sensitivity and reactivates apoptosis in TRAIL-resistant high-grade gliomas. In addition, our group showedthat bortezomib is able to sensitize TRAIL resistance by its proteasome inhibition independent effects in TRAIL-resistant HCC cells. Importantly, despite the fact that TRAIL can activate NF-κB (41, 42) and that bortezomib effectively inhibits NF-κB signaling, it seems that in HCC cells, inhibition of NF-κB may not be the major determinant of TRAIL sensitization by bortezomib as shown by our previous work (14, 17).

Numerous studies have shown that a combination of chemotherapy and targeted agents can enhance the antitumor activity of TRAIL and its agonists through cross talk between the intrinsic and extrinsic apoptotic pathways (8). Among the many novel agents with potential to sensitize or overcome TRAIL resistance, the combination of bortezomib and CS-1008 represents a fascinating strategy for several reasons. Bortezomib is approved for clinical use and exerts particularly tolerable toxicity when combined with other cytotoxic chemotherapeutic agents (such as doxorubicin, melphalan, or vincristine), and even when used in combination with intensive salvage chemotherapy in patients with refractory hematologic malignancies (43, 44). CS-1008, for its part, although still under clinical investigation, has shown an excellent toxicity profile with low hepatic toxicity (9, 10, 45), which is particularly suitable for patients with HCC. In addition, as CS-1008 is DR5 specific, the potency of CS-1008 may not be affected by problems with DR4 mutations or dysfunction. DR5 mutations have been reported to be infrequent in HCC cells (46). However, the exact machinery by which bortezomib downregulates CIP2A is still unknown. It is possible that bortezomib affects the transcription or translation of CIP2A through an as yet unidentified mechanism or affects the degradation of CIP2A at posttranslational level. Future work is needed in this area.

In summary, bortezomib sensitizes HCC cells to CS-1008–induced apoptosis through inhibiting a novel oncoprotein phosphatase interactive mechanism, the CIP2A-PP2A-p-Akt framework, and CIP2A may be a potential molecular target for HCC treatment. Moreover, combination of bortezomib, an agent with multiple cellular targets, and a specific anti-DR5 TRAIL agonist (CS-1008) is a promising anti-HCC–targeted therapy that warrants clinical trials. Future studies detailing the clinical role of CIP2A in HCC, and the machinery by which bortezomib affects CIP2A expression may lead to further progress in the development of molecular-targeted therapy for HCC.

A-L. Cheng is a consultant for Daiichi-Sankyo.

This study was supported by grants NTUH99-P04 from National Taiwan University Hospital (K-F. Chen), V99-B1-016 from Taipei Veterans General Hospital (C-Y. Liu), NSC98-2314-B-002-067-MY3 (K-F. Chen), and NSC99-2314-B-002-017-MY2 (K-F. Chen) from the National Science Council, Taiwan.

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.

1.
Verslype
C
,
Van Cutsem
E
,
Dicato
M
,
Arber
N
,
Berlin
JD
,
Cunningham
D
, et al
The management of hepatocellular carcinoma. Current expert opinion and recommendations derived from the 10th World Congress on Gastrointestinal Cancer, Barcelona, 2008
.
Ann Oncol
2009
;
20
Suppl 7
:
vii1
6
.
2.
Farazi
PA
,
DePinho
RA
. 
Hepatocellular carcinoma pathogenesis: from genes to environment
.
Nat Rev Cancer
2006
;
6
:
674
87
.
3.
Tanaka
S
,
Arii
S
. 
Molecularly targeted therapy for hepatocellular carcinoma
.
Cancer Sci
2009
;
100
:
1
8
.
4.
Llovet
JM
,
Ricci
S
,
Mazzaferro
V
,
Hilgard
P
,
Gane
E
,
Blanc
JF
, et al
Sorafenib in advanced hepatocellular carcinoma
.
N Engl J Med
2008
;
359
:
378
90
.
5.
Johnstone
RW
,
Frew
AJ
,
Smyth
MJ
. 
The TRAIL apoptotic pathway in cancer onset, progression and therapy
.
Nat Rev Cancer
2008
;
8
:
782
98
.
6.
Wang
S
,
El-Deiry
WS
. 
TRAIL and apoptosis induction by TNF-family death receptors
.
Oncogene
2003
;
22
:
8628
33
.
7.
Rowinsky
EK
. 
Targeted induction of apoptosis in cancer management: the emerging role of tumor necrosis factor-related apoptosis-inducing ligand receptor activating agents
.
J Clin Oncol
2005
;
23
:
9394
407
.
8.
Wiezorek
J
,
Holland
P
,
Graves
J
. 
Death receptor agonists as a targeted therapy for cancer
.
Clin Cancer Res
2010
;
16
:
1701
8
.
9.
Yada
A
,
Yazawa
M
,
Ishida
S
,
Yoshida
H
,
Ichikawa
K
,
Kurakata
S
, et al
A novel humanized anti-human death receptor 5 antibody CS-1008 induces apoptosis in tumor cells without toxicity in hepatocytes
.
Ann Oncol
2008
;
19
:
1060
7
.
10.
Saleh
MN
,
Percent
I
,
Wood
TE
,
Posey
J
 III
,
Shah
J
,
Carlisle
R
, et al
A phase I study of CS-1008 (humanized monoclonal antibody targeting death receptor 5 or DR5), administered weekly to patients with advanced solid tumors or lymphomas
.
J Clin Oncol
2008
;
26
:
3537
.
11.
Shin
EC
,
Seong
YR
,
Kim
CH
,
Kim
H
,
Ahn
YS
,
Kim
K
, et al
Human hepatocellular carcinoma cells resist to TRAIL-induced apoptosis, and the resistance is abolished by cisplatin
.
Exp Mol Med
2002
;
34
:
114
22
.
12.
Zhang
L
,
Fang
B
. 
Mechanisms of resistance to TRAIL-induced apoptosis in cancer
.
Cancer Gene Ther
2005
;
12
:
228
37
.
13.
Pathil
A
,
Armeanu
S
,
Venturelli
S
,
Mascagni
P
,
Weiss
TS
,
Gregor
M
, et al
HDAC inhibitor treatment of hepatoma cells induces both TRAIL-independent apoptosis and restoration of sensitivity to TRAIL
.
Hepatology
2006
;
43
:
425
34
.
14.
Chen
KF
,
Yeh
PY
,
Hsu
C
,
Hsu
CH
,
Lu
YS
,
Hsieh
HP
, et al
Bortezomib overcomes tumor necrosis factor-related apoptosis-inducing ligand resistance in hepatocellular carcinoma cells in part through the inhibition of the phosphatidylinositol 3-kinase/Akt pathway
.
J Biol Chem
2009
;
284
:
11121
33
.
15.
Orlowski
RZ
,
Kuhn
DJ
. 
Proteasome inhibitors in cancer therapy: lessons from the first decade
.
Clin Cancer Res
2008
;
14
:
1649
57
.
16.
Voorhees
PM
,
Orlowski
RZ
. 
The proteasome and proteasome inhibitors in cancer therapy
.
Annu Rev Pharmacol Toxicol
2006
;
46
:
189
213
.
17.
Chen
KF
,
Yeh
PY
,
Yeh
KH
,
Lu
YS
,
Huang
SY
,
Cheng
AL
. 
Down-regulation of phospho-Akt is a major molecular determinant of bortezomib-induced apoptosis in hepatocellular carcinoma cells
.
Cancer Res
2008
;
68
:
6698
707
.
18.
Chen
KF
,
Yu
HC
,
Liu
TH
,
Lee
SS
,
Chen
PJ
,
Cheng
AL
. 
Synergistic interactions between sorafenib and bortezomib in hepatocellular carcinoma involve PP2A-dependent Akt inactivation
.
J Hepatol
2009
;
52
:
88
95
.
19.
Janssens
V
,
Goris
J
. 
Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling
.
Biochem J
2001
;
353
:
417
39
.
20.
Xu
Y
,
Xing
Y
,
Chen
Y
,
Chao
Y
,
Lin
Z
,
Fan
E
, et al
Structure of the protein phosphatase 2A holoenzyme
.
Cell
2006
;
127
:
1239
51
.
21.
Neviani
P
,
Santhanam
R
,
Trotta
R
,
Notari
M
,
Blaser
BW
,
Liu
S
, et al
The tumor suppressor PP2A is functionally inactivated in blast crisis CML through the inhibitory activity of the BCR/ABL-regulated SET protein
.
Cancer Cell
2005
;
8
:
355
68
.
22.
Junttila
MR
,
Puustinen
P
,
Niemelä
M
,
Ahola
R
,
Arnold
H
,
Böttzauw
T
, et al
CIP2A inhibits PP2A in human malignancies
.
Cell
2007
;
130
:
51
62
.
23.
Soo
Hoo L
,
Zhang
JY
,
Chan
EK
. 
Cloning and characterization of a novel 90 kDa ‘companion' auto-antigen of p62 overexpressed in cancer
.
Oncogene
2002
;
21
:
5006
15
.
24.
Côme
C
,
Laine
A
,
Chanrion
M
,
Edgren
H
,
Mattila
E
,
Liu
X
, et al
CIP2A is associated with human breast cancer aggressivity
.
Clin Cancer Res
2009
;
15
:
5092
100
.
25.
Li
W
,
Ge
Z
,
Liu
C
,
Liu
Z
,
Björkholm
M
,
Jia
J
, et al
CIP2A is overexpressed in gastric cancer and its depletion leads to impaired clonogenicity, senescence, or differentiation of tumor cells
.
Clin Cancer Res
2008
;
14
:
3722
8
.
26.
Forero-Torres
A
,
Shah
J
,
Wood
T
,
Posey
J
,
Carlisle
R
,
Copigneaux
C
, et al
Phase I trial of weekly tigatuzumab, an agonistic humanized monoclonal antibody targeting death receptor 5 (DR5)
.
Cancer Biother Radiopharm
2010
;
25
:
13
9
.
27.
Okano
H
,
Shiraki
K
,
Inoue
H
,
Kawakita
T
,
Yamanaka
T
,
Deguchi
M
, et al
Cellular FLICE/caspase-8-inhibitory protein as a principal regulator of cell death and survival in human hepatocellular carcinoma
.
Lab Invest
2003
;
83
:
1033
43
.
28.
Eggert
A
,
Grotzer
MA
,
Zuzak
TJ
,
Wiewrodt
BR
,
Ho
R
,
Ikegaki
N
, et al
Resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in neuroblastoma cells correlates with a loss of caspase-8 expression
.
Cancer Res
2001
;
61
:
1314
9
.
29.
Kim
SH
,
Ricci
MS
,
El-Deiry
WS
. 
Mcl-1:a gateway to TRAIL sensitization
.
Cancer Res
2008
;
68
:
2062
4
.
30.
Fulda
S
,
Meyer
E
,
Debatin
KM
. 
Inhibition of TRAIL-induced apoptosis by Bcl-2 overexpression
.
Oncogene
2002
;
21
:
2283
94
.
31.
Zhang
XD
,
Zhang
XY
,
Gray
CP
,
Nguyen
T
,
Hersey
P
. 
Tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis of human melanoma is regulated by smac/DIABLO release from mitochondria
.
Cancer Res
2001
;
61
:
7339
48
.
32.
Bortul
R
,
Tazzari
PL
,
Cappellini
A
,
Tabellini
G
,
Billi
AM
,
Bareggi
R
, et al
Constitutively active Akt1 protects HL60 leukemia cells from TRAIL-induced apoptosis through a mechanism involving NF-kappaB activation and cFLIP(L) up-regulation
.
Leukemia
2003
;
17
:
379
89
.
33.
Ehrhardt
H
,
Fulda
S
,
Schmid
I
,
Hiscott
J
,
Debatin
KM
,
Jeremias
I
. 
TRAIL induced survival and proliferation in cancer cells resistant towards TRAIL-induced apoptosis mediated by NF-kappaB
.
Oncogene
2003
;
22
:
3842
52
.
34.
Hetschko
H
,
Voss
V
,
Seifert
V
,
Prehn
JH
,
Kogel
D
. 
Upregulation of DR5 by proteasome inhibitors potently sensitizes glioma cells to TRAIL-induced apoptosis
.
FEBS J
2008
;
275
:
1925
36
.
35.
Sayers
TJ
,
Brooks
AD
,
Koh
CY
,
Ma
W
,
Seki
N
,
Raziuddin
A
, et al
The proteasome inhibitor PS-341 sensitizes neoplastic cells to TRAIL-mediated apoptosis by reducing levels of c-FLIP
.
Blood
2003
;
102
:
303
10
.
36.
Feng
X
,
Yan
J
,
Wang
Y
,
Zierath
JR
,
Nordenskjöld
M
,
Henter
JI
, et al
The proteasome inhibitor bortezomib disrupts tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression and natural killer (NK) cell killing of TRAIL receptor-positive multiple myeloma cells
.
Mol Immunol
2010
:
47
:
2388
96
.
37.
Liu
J
,
Qu
XJ
,
Xu
L
,
Zang
Y
,
Qu
JL
,
Hou
KZ
, et al
Bortezomib synergizes TRAIL-induced apoptosis in gastric cancer cells
.
Dig Dis Sci
2010
;
55
:
3361
8
.
38.
Liu
X
,
Yue
P
,
Chen
S
,
Hu
L
,
Lonial
S
,
Khuri
FR
, et al
The proteasome inhibitor PS-341 (bortezomib) up-regulates DR5 expression leading to induction of apoptosis and enhancement of TRAIL-induced apoptosis despite up-regulation of c-FLIP and survivin expression in human NSCLC cells
.
Cancer Res
2007
;
67
:
4981
8
.
39.
Seki
N
,
Toh
U
,
Sayers
TJ
,
Fujii
T
,
Miyagi
M
,
Akagi
Y
, et al
Bortezomib sensitizes human esophageal squamous cell carcinoma cells to TRAIL-mediated apoptosis via activation of both extrinsic and intrinsic apoptosis pathways
.
Mol Cancer Ther
2010
;
9
:
1842
51
.
40.
Brooks
AD
,
Jacobsen
KM
,
Li
W
,
Shanker
A
,
Sayers
TJ
. 
Bortezomib sensitizes human renal cell carcinomas to TRAIL apoptosis through increased activation of caspase-8 in the death-inducing signaling complex
.
Mol Cancer Res
2010
;
8
:
729
38
.
41.
Franco
AV
,
Zhang
XD
,
Van Berkel
E
,
Sanders
JE
,
Zhang
XY
,
Thomas
WD
, et al
The role of NF-kappa B in TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis of melanoma cells
.
J Immunol
2001
;
166
:
5337
45
.
42.
Degli-Esposti
MA
,
Dougall
WC
,
Smolak
PJ
,
Waugh
JY
,
Smith
CA
,
Goodwin
RG
. 
The novel receptor TRAIL-R4 induces NF-kappaB and protects against TRAIL-mediated apoptosis, yet retains an incomplete death domain
.
Immunity
1997
;
7
:
813
20
.
43.
Dunleavy
K
,
Pittaluga
S
,
Czuczman
MS
,
Dave
SS
,
Wright
G
,
Grant
N
, et al
Differential efficacy of bortezomib plus chemotherapy within molecular subtypes of diffuse large B-cell lymphoma
.
Blood
2009
;
113
:
6069
76
.
44.
Messinger
Y
,
Gaynon
P
,
Raetz
E
,
Hutchinson
R
,
Dubois
S
,
Glade-Bender
J
, et al
Phase I study of bortezomib combined with chemotherapy in children with relapsed childhood acute lymphoblastic leukemia (ALL): a report from the therapeutic advances in childhood leukemia (TACL) consortium
.
Pediatr Blood Cancer
2010
;
55
:
254
9
.
45.
Sung
ES
,
Park
KJ
,
Lee
SH
,
Jang
YS
,
Park
SK
,
Park
YH
, et al
A novel agonistic antibody to human death receptor 4 induces apoptotic cell death in various tumor cells without cytotoxicity in hepatocytes
.
Mol Cancer Ther
2009
;
8
:
2276
85
.
46.
Jeng
YM
,
Hsu
HC
. 
Mutation of the DR5/TRAIL receptor 2 gene is infrequent in hepatocellular carcinoma
.
Cancer Lett
2002
;
181
:
205
8
.