Aggresome Disruption: a Novel Strategy to Enhance Bortezomib- Induced Apoptosis in Pancreatic Cancer Cells

Research Article

Aggresome Disruption: A Novel Strategy to Enhance Bortezomib- Induced Apoptosis in Pancreatic Cancer Cells

Steffan T. Nawrocki,1 Jennifer S. Carew,2 Maria S. Pino,3,7 Ralph A. Highshaw,4 Robert H.I. Andtbacka,5 Kenneth Dunner, Jr.,1 Ashutosh Pal,6 William G. Bornmann,6 Paul J. Chiao,5 Peng Huang,2 Henry Xiong,3 JamesL. Abbruzzese, 3 and David J. McConkey1

Departments of 1Cancer Biology, 2Molecular Pathology, 3Gastrointestinal Medical Oncology, 4Urology, 5Surgical Oncology, and 6Experimental Diagnostic Imaging, The University of Texas M. D. Anderson Cancer Center, Houston, Texas and 7Division of Medical Oncology ‘‘A,’’ Regina Elena Cancer Institute, Rome, Italy

Abstract inhibition of NF-nB activity accounts for only a small fraction of the anticancer activity of bortezomib (8, 9), indicating that the The proteasome inhibitor bortezomib (formerly known as PS-341) recently received Food and Drug Administration mechanisms of action of the drug remain to be fully approval for the treatment of multiple myeloma, and its characterized. activity is currently being evaluated in solid tumors. Bortezo- We suspected that bortezomib induces cell death by causing a mib triggers apoptosis in pancreatic cancer cells, but the buildup of misfolded and otherwise damaged proteins, thereby mechanisms involved have not been fully elucidated. Here, we triggering endoplasmic reticulum (ER) stress (10–13). The ER is show that pancreatic cancer cells exposed to bortezomib formed especially vulnerable to the presence of misfolded proteins because aggregates of ubiquitin-conjugated proteins (‘‘aggresomes’’) of its involvement in post-translational modification, folding and in vitro and in vivo. Bortezomib-induced aggresome formation assembly of newly synthesized proteins, and regulated calcium was determined to be cytoprotective and could be disrupted storage (14). Pancreatic epithelial cells possess a highly developed using histone deacetylase (HDAC) 6 small interfering RNA or ER due to a heavy engagement in insulin and digestive enzyme chemical HDAC inhibitors, which resulted in endoplasmic secretion (15), and they seem to be particularly sensitive to ER reticulum stress and synergistic levels of apoptosis in vitro and stress-induced apoptosis (16, 17). In previous studies, we showed that bortezomib stimulates in an orthotopic pancreatic cancer xenograft model in vivo. Interestingly, bortezomib did not induce aggresome formation apoptosis in approximately half of the tested human pancreatic in immortalized normal human pancreatic epithelial cells cancer cell lines (18, 19). Here, we show that bortezomib causes the in vitro or in murine pancreatic epithelial cells in vivo. In ad- sequestration of ubiquitin-conjugated proteins into structures dition, these cells did not undergo apoptosis following termed aggresomes in these cells. Aggresomes seem to participate treatment with bortezomib, suberoylanilide hydroxamic acid, in a cytoprotective response that is activated in response to or the combination, showing tumor selectivity. Taken together, proteasome inhibition perhaps by shuttling ubiquitylated proteins our study shows that inhibition of aggresome formation can to lysosomes for degradation (20). Previous studies showed that strongly potentiate the efficacy of bortezomib and provides the bortezomib interacts synergistically with histone deacetylase foundation for clinical trials of bortezomib in combination (HDAC) inhibitors to induce apoptosis in multiple myeloma and chronic myelogenous leukemia (21–23). We show that these effects with HDAC inhibitors for the treatment of pancreatic cancer. are closely associated with disruption of aggresome formation (Cancer Res 2006; 66(7): 3773-81) leading to enhanced ER stress. Together, the results strongly suggest that bortezomib-induced apoptosis is mediated via ER Introduction stress, and they identify a clinically viable strategy (disruption of The 26S proteasome is responsible for the degradation of aggresome formation) to overcome bortezomib resistance in a >80% of all cellular proteins (1, 2) and plays a key regulatory role subset of human pancreatic cancer cells. in cell cycle progression and apoptosis, making it an attractive therapeutic target in cancer (3, 4). Bortezomib (Velcade; formerly Materials and Methods known as PS-341) is a peptide boronate inhibitor of the proteasome that recently received Food and Drug Administration Animals and Cell Lines (FDA) approval for the treatment of multiple myeloma (5), and Male nude mice (BALB/c background) were purchased from the it is currently being evaluated for the treatment of solid tumors American Production Area of the National Cancer Institute (NCI)- Frederick Cancer Research and Development Center (Frederick, MD). (6). The antineoplastic effects of bortezomib have been n L3.6pl human pancreatic cancer cells were established as described attributed, in part, to inhibition of I B degradation leading to previously (24). All other pancreatic cancer cell lines were obtained from inactivation of the prosurvival transcription factor, nuclear the American Type Culture Collection (Rockville, MD). Human pancreatic factor-nB (NFnB; ref. 7). However, recent findings showed that cancer cell lines were maintained in MEM. The immortal pancreatic duct epithelium cell line HPDE6-E6E7 was established and cultured as described previously (25, 26).

Requests for reprints: David J. McConkey, Department of Cancer Biology, The Antibodies and Chemicals University of Texas M. D. Anderson Cancer Center, Box 173, 1515 Holcombe Boulevard, Antibodies were obtained from the following commercial sources: anti- Houston, TX 77030. Phone: 713-792-8591; Fax: 713-792-8747; E-mail: dmcconke@ active caspase-3 (Transduction Laboratories, San Diego, CA); anti-HDAC6, mdanderson.org. I2006 American Association for Cancer Research. HDAC7, and HDAC1 (Cell Signaling, Beverly, MA); anti-ubiquitin (Santa doi:10.1158/0008-5472.CAN-05-2961 Cruz Biotechnology, Santa Cruz, CA); and anti-calreticulin (StressGen, www.aacrjournals.org 3773 Cancer Res 2006; 66: (7). April 1, 2006

Confocal Microscopy Cell lines. Pancreatic cancer and normal pancreatic epithelial cells were plated on chamber slides before drug exposure. Cells were fixed with 4% paraformaldehyde, permeabilized using 0.2% Triton X-100, and incubated overnight with indicated primary antibodies. Fluorescent secondary antibodies were used to visualize protein localization. ToPro- 3 (Molecular Probes) was used to counterstain the nucleus. Images were obtained using a Zeiss LSM510 confocal microscope (Oberkochem, Germany). Tumor sections. Paraffin sections were prepared as described previously (19). Sections were incubated overnight with anti-ubiquitin antibody followed by 1-hour incubation with Alexa Fluor 488 secondary antibody. Analysis of DNA fragmentation by terminal deoxynucleotidyl transferase– mediated nick end labeling (TUNEL) was done using a commercial kit (Promega, Madison, WI). PI was used to counterstain the nucleus. All slides were mounted using Prolong anti-fade reagent (Molecular Probes). Images were obtained using confocal microscopy as described above. Percentages of TUNEL-positive cells were determined using a laser scanning cytometer (LSC) as described previously (19). Transmission Electron Microscopy Transmission electron microscopy (TEM) of cells was done as described previously (27). Ultrathin sections were cut in a LKB Ultracut microtome (Leica, Deerfield, IL), stained with uranyl acetate and lead citrate in a LKB Ultrostainer (Leica), and examined in a JEM 1010 TEM (JEOL, Inc., Peabody, MA) at an accelerating voltage of 80 kV. Digital images were obtained using the AMT Imaging System (Advanced Microscopy Techniques Corp., Danvers, MA). Immunoblotting Cells (1 Â 105) were incubated with 100 nmol/L bortezomib for 24 hours. Cells were collected using a cell scraper at 4jC and lysed as described previously (19). Total cellular protein (f25 Ag) from each sample was subjected to SDS-PAGE, proteins were transferred to nitrocellulose membranes, and the membranes were blocked with 5% nonfat milk in a TBS solution containing 0.1% Tween 20 for 1 hour. The blots were then probed overnight with relevant antibodies, washed, and probed with species-specific secondary antibodies coupled to HRP. Immunoreactive material was detected by enhanced chemiluminescence (West Pico Pierce, Inc., Rockville, IL). Measurement of Intracellular Ca2+ Levels Cells were grown in medium with or without 100 nmol/L bortezomib, 5 Amol/L SAHA, or a combination of the two drugs for 12 hours. Cells were collected, washed in PBS, and incubated with 1 Amol/L Calcium Green-1 acetoxymethyl ester (Molecular Probes) for 30 minutes. Flow cytometric analysis of stained cells was done with a Becton-Dickinson FACSCalibur Figure 1. Bortezomib treatment induces ubiquitin-conjugated protein (San Jose, CA). accumulation and aggresome formation. A, bortezomib (BZ) stabilizes ubiquitylated proteins (Ub-proteins). L3.6pl cells were incubated with 100 nmol/L Preparation and Transfection of Small Interfering RNA bortezomib for 24 hours. Immunoblotting was done with an anti-ubiquitin antibody as described in Materials and Methods. B, bortezomib induces The annealed double-stranded HDAC6 and luciferase small interfering aggresome formation. L3.6pl cells were incubated for 24 hours with 100 nmol/L RNA (siRNA) were obtained from Dharmacon (Lafayette, CO): HDAC6 bortezomib and stained for detection of ubiquitin (Ub) and calreticulin as siRNA sense 5V-GCAGUUAAAUGAAUUCCAUUU-3V and antisense 5V-P- described in Materials and Methods. Fluorescence was visualized by confocal AUGGAAUUCAUUUAACUGCUU-3V. For control, siRNA directed against microscopy. C, TEM reveals aggresome structure. L3.6pl cells were fixed and prepared for TEM as described in Materials and Methods. D, bortezomib does firefly luciferase was used. Cells were transfected with 100 nmol/L of the not induce aggresome formation in immortalized normal human pancreatic above siRNA using Oligofectamine (Invitrogen, Carlsbad, CA) according to epithelial cells (HPDE6-E6E7). Cells were incubated with 100 nmol/L bortezomib the manufacturer’s protocol. Transfected cells were incubated at 37jC for for 24 hours, and aggresome formation was assessed by anti-ubiquitin 40 hours without changing the medium. Efficiency of RNA interference immunofluorescence as described above. (RNAi) was measured by immunoblotting and immunocytochemistry using anti-HDAC6 antibodies. Victoria, British Columbia, Canada). Horseradish peroxidase (HRP)– conjugated secondary antibodies for immunoblotting were obtained from Quantification of DNA Fragmentation Amersham Pharmacia Biotech (Piscataway, NJ). Alexa Fluor 488 goat anti- DNA fragmentation was measured by PI staining and fluorescence- mouse antibody and Texas red goat anti-rabbit antibody were obtained activated cell sorting (FACS) analysis as described previously (19). Cells from Molecular Probes (Eugene, OR). Bortezomib was kindly provided by were plated in six-well plates (1 Â 106 per well). Following drug Millennium Pharmaceuticals (Boston, MA). Propidium iodide (PI) and incubation, cells were harvested, pelleted by centrifugation, and trichostatin A were obtained from Sigma Chemical (St. Louis, MO). resuspended in PBS containing 50 Ag/mL PI, 0.1% Triton X-100, and Suberoylanilide hydroxamic acid (SAHA) was synthesized by A. Pal and 0.1% sodium citrate. DNA fragmentation was quantified by flow W.G. Bornmann (M. D. Anderson Cancer Center, Houston, TX). cytometric analysis.

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Quantification of Apoptotic Cells and Aggresome Formation particles in a central core (Fig. 1C). Although bortezomib induced Aggresome formation was detected by immunofluorescence staining aggresome accumulation in most cancer cells, it did not induce with an anti-ubiquitin antibody and confocal microscopy. The presence aggresomes in immortalized normal pancreatic epithelial cells of a single perinuclear inclusion was considered indicative of aggresome (Fig. 1D). formation, and the results were confirmed by TEM. Aggresomes were quantified manually by scoring f200 cells as aggresome positive or aggresome negative in three separate fields by confocal microscopy. Cell death was determined in the same cell populations by immunofluorescence with an antibody specific for active caspase-3 or by fluorescent TUNEL, which were done simultaneously with visualization of aggresomes to distinguish aggresome-positive and aggresome-negative apoptotic cells. ToPro-3 was used to counterstain nuclei. To quantify apoptotic cells with or without aggresomes, f200 apoptotic cells were counted and scored as aggresome positive (defined aggresome) or aggresome negative (no visual aggresome) using a confocal microscope, and scoring was repeated thrice. Orthotopic Implantation of Tumor Cells and Treatment Schedule L3.6pl pancreatic cancer cells were harvested from culture flasks after brief trypsinization and transferred to serum-free HBSS. Only single-cell suspensions of >90% viability determined by trypan blue exclusion were used. Male nude mice were anesthetized with methoxyflurane, a small left abdominal flank incision was made, and tumor cells (1 Â 106) were injected into the subcapsular region of the pancreas using a 30-gauge needle and a calibrated push button-controlled dispensing device (Hamilton Syringe Co., Reno, NV). A successful subcapsular intrapancreatic injection of tumor cells was confirmed by the appearance of a fluid bubble without i.p. leakage. To further prevent leakage, a cotton swab was held cautiously for 1 minute over the site of injection. The abdominal wound was closed in one layer with wound clips (Autoclip; Clay Adams, Parsippany, NJ). Tumors were established for 14 days before therapy. Animals were then injected i.p. with bortezomib at a dosage of 1 mg/kg every 72 hours, 50 mg/ kg SAHA daily, or a combination of the two drugs for 21 days. Mice were killed by cervical dislocation, and primary tumors in the pancreas were excised and weighed. For immunohistochemistry, tumor tissue was formalin fixed and paraffin embedded. Statistical Analyses Statistical significance of differences observed in drug-treated and control samples were determined using the Tukey-Kramer comparison test. Differences were considered significant in all experiments at P < 0.05.

Results Bortezomib stimulates aggresome formation in pancreatic cancer cells. Inhibition of the proteasome resulted in the accumulation of ubiquitylated proteins in human pancreatic cancer cells as assessed by immunoblotting (Fig. 1A). Analysis of the subcellular distribution of these ubiquitin conjugates by immunoflourescent anti-ubiquitin staining and confocal microscopy revealed that they formed large perinuclear structures consistent in appearance with aggresomes (Fig. 1B; ref. 13). TEM revealed that the aggresomes were organized as electron-dense

Figure 2. Aggresome formation protects against bortezomib-induced apoptosis. A, bortezomib-treated L3.6pl cells that fail to form aggresomes exhibit ER dilation. Cells were prepared as described previously, and representative images were taken of the ER structure within the drug-treated cells. B and C, aggresome-positive cells are resistant to bortezomib-induced apoptosis. L3.6pl cells were treated with 100 nmol/L bortezomib for 24 hours. Cells were stained with anti-ubiquitin antibody to visualize aggresomes, and either anti-active caspase-3 antibody or TUNEL assay was used to distinguish apoptotic cells. ToPro-3 was used to counterstain the nucleus in (B). Representative images. D, quantification of apoptotic cells with or without aggresomes. Apoptotic cells (f200) were counted and scored as aggresome positive (single-defined aggresome) or aggresome negative (no visual aggresome) using a confocal microscope. Columns, mean (n = 3); bars, SD. www.aacrjournals.org 3775 Cancer Res 2006; 66: (7). April 1, 2006

Figure 3. SAHA disrupts bortezomib-induced aggresome formation and sensitizes aggresome-positive cells to apoptosis. A, majority (6 of 8) of pancreatic cancer cell lines form a significant percentage of aggresomes following bortezomib treatment, which can be inhibited by SAHA. Cells were treated with 100 nmol/L bortezomib with or without 5 Amol/L SAHA for 24 hours, stained with anti-ubiquitin antibody to visualize aggresomes, and quantified using confocal microscopy. Cells (f200) were counted and scored per cell line. Columns, mean (n = 3); bars, SD. B, kinetic analysis of aggresome formation. Panc1and L3.6pl cells were treated with 100 nmol/L bortezomib with or without 5 Amol/L SAHA for the indicated times. The presence of a single perinuclear inclusion was scored as an aggresome. Quantification was done as described in Materials and Methods. C, HDAC inhibitors block aggresome formation. Cells were treated with 100 nmol/L bortezomib and 5 Amol/L SAHA. Green, ubiquitin-conjugated proteins were visualized using Alexa Fluor 488 secondary antibody; blue, nucleus was counterstained with ToPro-3. Image was generated by confocal microscopy and is representative of the treatment group. Similar results were obtained when trichostatin A was used instead of SAHA (data not shown). TEM of drug-exposed L3.6pl cells. Arrows, ubiquitin-conjugated proteins. Note the single perinuclear aggresome in bortezomib-treated cells and the dispersion of ubiquitin-conjugated proteins in cells exposed to bortezomib + SAHA. D, HDAC inhibitors enhance bortezomib-induced apoptosis in aggresome-forming cells. Left, cells were incubated with 100 nmol/L bortezomib, 500 nmol/L trichostatin A (TSA), 5 Amol/L SAHA, or combinations for 24 hours. Apoptosis was measured by PI-FACS analysis. Columns, mean (n = 3); bars, SD. Right, immortalized normal pancreatic epithelial cells (HPDE6-E6E7) are unaffected by the bortezomib and SAHA combination. Cells were treated with 100 nmol/L bortezomib, 5 Amol/L SAHA, or combination for 24 hours, and apoptosis was measured by PI-FACS analysis. Columns, mean (n = 3); bars, SD.

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negative cells appeared more like cells exposed to thapsigargin (an ER Ca2+ ATPase inhibitor that is known to induce ER stress) and displayed significant dilation of the ER (Fig. 2A). To further investigate the relationship between aggresome formation and cell death, we quantified the levels of apoptosis in aggresome- positive and aggresome-negative cells by confocal microscopy (Fig. 2B and C). Almost all of the aggresome-positive cells were TUNEL and active-caspase-3 negative, whereas aggresome- negative cells were TUNEL and active-caspase-3 positive (Fig. 2D). Thus, aggresomes seem to play a cytoprotective function that limits bortezomib-induced apoptosis. HDAC inhibition disrupts aggresomes and enhances bortezomib-induced apoptosis. We next determined the prevalence of

Figure 4. HDAC6 inhibition disrupts aggresome formation and sensitizes pancreatic cancer cells to bortezomib-induced apoptosis. A, HDAC6 knockdown disrupts aggresome formation. RNAi was done as described in Materials and Methods. Cells were stained with anti-ubiquitin antibody to visualize aggresomes and anti-HDAC6 antibody to examine expression levels. Images were obtained by confocal microscopy. Quantification of aggresome-positive cells. Cells (f200) were counted and scored as aggresome positive (single-defined aggresome) or aggresome negative (no visible aggresome) using a confocal microscope. Columns, mean (n = 3); bars, SD. B, knockdown of HDAC6 enhances bortezomib-induced apoptosis. L3.6pl cells were treated with 100 nmol/L bortezomib in the presence or absence of HDAC6 siRNA. Luciferase siRNA was used as an off-target control. Immunoblotting showed silencing of HDAC6 (H6) without change in HDAC7 (H7) or HDAC1( H1) expression. Apoptosis was determined by PI-FACS. Columns, mean (n = 3); bars, SD. Asterisk, significant difference from bortezomib-treated untransfected (UN) and luciferase (LUC) siRNA-transfected cells. Figure 5. Aggresome disruption sensitizes pancreatic cancer cells to ER stress. A, electron micrographs of ER morphology in cells exposed to 100 nmol/L Aggresome formation limits bortezomib-induced apoptosis. bortezomib, 5 Amol/L SAHA, or both drugs. Black arrows, ER; red arrows, aggresome in bortezomib-treated cells or disrupted aggresome in combination- We next investigated the relationship between aggresome treated cells. B, exposure to bortezomib + SAHA disrupts intracellular Ca2+ formation and bortezomib-induced ER stress and apoptosis. homeostasis. L3.6pl cells were treated for 12 hours with 100 nmol/L bortezomib, Visual examination of electron micrographs of bortezomib- 5 Amol/L SAHA, or both drugs, loaded with Calcium Green-1acetoxymethyl ester, and analyzed by flow cytometry as described in Materials and Methods. treated L3.6pl cells indicated that aggresome-positive cells Columns, mean (n = 3); bars, SD. Asterisk, significant difference from either displayed no visible signs of ER stress, whereas aggresome- bortezomib or SAHA single-agent treatment. www.aacrjournals.org 3777 Cancer Res 2006; 66: (7). April 1, 2006

Figure 6. Bortezomib induces aggresome formation in an orthotopic pancreatic cancer model, and SAHA enhances its antitumor activity. A, left, bortezomib induces aggresome formation in orthotopic L3.6pl tumors in vivo but not in normal murine pancreatic epithelial cells. Right, SAHA disrupts aggresome formation in vivo. Green, tissue sections were stained with anti-ubiquitin antibody followed by Alexa Fluor 488 fluorescent secondary antibody to visualize aggresomes; red, PI was used to counterstain the nucleus. Four random sections were selected, and the percentage of aggresome-positive cells was counted by confocal microscopy. Columns, mean; bars, SD. B, combination therapy with bortezomib + SAHA produces a significant increase in tumor cell apoptosis in vivo. Green, apoptosis was measured by TUNEL staining of tissue sections; red, tissues were counterstained with PI to detect cell nuclei. TUNEL-positive cells were quantified using LSC as described in Materials and Methods. Columns, mean (n = 4); bars, SD. Asterisk, significant difference compared with levels observed with either single agent. C, schematic representation of the mechanisms underlying apoptosis induced by bortezomib + SAHA. By inhibiting proteasome-mediated protein degradation, bortezomib promotes the accumulation and subsequent aggregation of ubiquitin-conjugated proteins leading to proteotoxicity. These protein aggregates can be sequestered in a cytoprotective aggresomes via mechanisms that require HDAC6 activity. SAHA-mediated inhibition of HDAC6 disrupts aggresome formation, yielding toxic microaggregates that enhance proteotoxicity, ER stress, and apoptosis.

bortezomib-induced aggresome formation in a panel of eight detected in immortalized normal pancreatic epithelial cells pancreatic cancer cell lines. Six of them, including two bortezomib- (Fig. 1D; data not shown). Kinetic analysis of aggresome formation resistant cell lines, displayed significant levels of aggresome in Panc1 (bortezomib-resistant) and L3.6pl (bortezomib-sensitive) formation in >40% of cells, which could be inhibited by the HDAC pancreatic cancer cells showed that 60% to 70% of cells possessed inhibitor, SAHA (Fig. 3A). Interestingly, no aggresomes were an aggresome by 12 hours after exposure to 100 nmol/L

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2006 American Association for Cancer Research. Inhibition of HDAC6 Enhances Bortezomib-Induced Death bortezomib (Fig. 3B). The HDAC inhibitors SAHA and trichostatin systemic therapy of bortezomib in combination with SAHA is well A blocked bortezomib-induced aggresome formation (Fig. 3B tolerated, produces the desired effects on aggresome formation and C) and dramatically sensitized aggresome-positive cells to and cell death, and displays selectivity for tumor cells. bortezomib-induced apoptosis (Fig. 3D). Because the immortalized normal pancreatic epithelial cells did not form aggresomes, they were unaffected by the combination of bortezomib and SAHA Discussion (Fig. 3D). Because previous work implicated the cytosolic HDAC6 The proteasome has become an attractive target for pharmaco- in proteasome inhibitor-mediated aggresome formation in other logic inhibition in cancer. Bortezomib is the first proteasome cellular models (28, 29), we tested the effects of silencing HDAC6 inhibitor evaluated in clinical trials, where it displayed manageable expression on aggresome formation and apoptosis. Consistent with toxicity at target doses in a variety of different tumor types (31) and the previous results (28, 29), silencing HDAC6 disrupted aggresome received FDA approval in 2003 for use in the treatment of multiple formation (Fig. 4A) and sensitized L3.6pl pancreatic cancer cells to myeloma (5). In vitro and in vivo studies done by our laboratory and bortezomib-induced apoptosis (Fig. 4B). Because aggresomes others have shown that bortezomib also has promising antitumor seemed to segregate with viable cells, we examined the effects of activity in pancreatic cancer models (18, 19, 32, 33). Bortezomib disruption on ER stress. The combination of bortezomib plus activates the mitochondrial (intrinsic) pathway of apoptosis SAHA produced a significant increase in ER dilation (Fig. 5A) and (31, 34, 35), but recent evidence indicates that these effects may ER Ca2+ release (Fig. 5B; ref. 30) compared with the levels observed occur downstream of events initiated within the ER (36, 37). in cells exposed to either single agent. Here, we show that pancreatic cancer cells exposed to Effects of bortezomib and SAHA in orthotopic human bortezomib contain electron-dense structures consisting of ubiq- pancreatic tumors. In a final series of experiments, we uitin-conjugated proteins that have been termed aggresomes (38). investigated the effects of bortezomib with or without SAHA on In pancreatic cancer cells, aggresomes seem to play a cytopro- aggresome formation and cell death in orthotopic L3.6pl tumors. tective function presumably sequestering aggregated ubiquitylated Established tumors were treated biweekly with 1 mg/kg bortezo- proteins and perhaps targeting them for lysosomal degradation mib, daily with 50 mg/kg SAHA, or a combination of the two agents (20), thereby limiting ER stress (Fig. 5; ref. 13). Aggresome for 21 days, and the experiment was done twice. Tumors treated formation requires the expression of a cytosolic, cytoskeleton- with bortezomib alone contained numerous aggresomes that were associated HDAC (HDAC6) as shown by our observation that disrupted in tumors treated with bortezomib plus SAHA (Fig. 6A). aggresomes are abolished in cells transfected with a HDAC6- Importantly, bortezomib did not induce aggresomes in adjacent specific siRNA construct (Fig. 4A). Our data are consistent with a normal murine pancreatic epithelial cells (Fig. 6A). Therapy with previous study, which showed that HDAC6 silencing or expression either bortezomib or SAHA alone stimulated moderate increases in of enzymatically inactive HDAC6 proteins prevented aggresome tumor cell apoptosis as measured by TUNEL staining and LSC formation in a neuronal model system (24). Virtually all analysis, whereas combination therapy led to much higher levels of bortezomib-induced apoptosis was restricted to aggresome-nega- cell death (Fig. 6B). No significant numbers of TUNEL-positive tive cells, and agents that disrupted them (HDAC6 siRNA or normal epithelial cells were observed following any of these chemical HDAC inhibitors) dramatically increased cell death, treatments (data not shown). Finally, combination therapy reversing bortezomib resistance in two of the cell lines (Figs. 3 significantly reduced pancreatic tumor weight compared with and 4). Importantly, a more selective chemical inhibitor of HDAC6 therapy with either agent alone (Table 1). Importantly, gastroin- (tubacin) was isolated by Schreiber’s laboratory (39), and in a more testinal toxicity (diarrhea) and weight loss were minimal and linked recent study, Anderson’s group showed that it also synergized with to the effects of bortezomib (Table 1). These data show that bortezomib to induce apoptosis in multiple myeloma cells (29).

Table 1. Therapy of L3.6pl human pancreatic cancer cells implanted into the pancreas of nude mice

Experiment no. Treatment group Tumor Tumor Tumor Body Mortality Gastrointestinal incidence weight (g) volume (mm3) weight (g) toxicity

1 Control 10/10 1.6 902 25.1 0 0/10 Bortezomib 10/10 0.9 526 23.5 0 6/10 SAHA 10/10 1.0 688 25.5 0 0/10 Bortezomib + SAHA 10/10 0.4* 253* 23.4 0 5/10 2 Control 10/10 1.3 763 24.8 0 0/10 Bortezomib 10/10 0.7 432 23 0 5/10 SAHA 10/10 1.1 655 25.3 0 0/10 Bortezomib + SAHA 10/10 0.4* 260* 23.3 0 5/10

NOTE: L3.6pl cells were injected into the pancreas of nude mice and established for 14 days. Mice were injected i.p. with DMSO and saline (control), bortezomib (1 mg/kg) every 72 hours, SAHA (50 mg/kg) daily, or both drugs for 21 days. Following 3 weeks of treatment, mice were sacrificed, and tumor weight, tumor volume, and body weight were recorded. All mice had pancreatic tumors. No mice mortality occurred during either experiment. Gastrointestinal toxicity indicates diarrhea as characterized by loose stools in the bowel. *P < 0.05versus control, bortezomib, and SAHA treatment groups.

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Therefore, it will be of interest to compare tubacin to the more observations, orthotopic implanted MiaPaCa-2 tumors in mice conventional pan-HDAC inhibitors with respect to its effects on treated with bortezomib also did not form aggresomes (data not bortezomib-induced aggresome formation and apoptosis in shown). Molecular chaperones interact with nonnative protein preclinical models of human pancreatic cancer. conformations to block protein aggregation and toxicity. It is Aggresomes have been observed in many pathologic states, possible that the failure of the MiaPaCa-2 and Hs766T cells to form including neurodegenerative diseases, such as Alzheimer’s disease aggresomes was due to high chaperone [heat-shock protein (Hsp)] and Parkinson’s disease (11, 40). A recent report showed that cells levels.9 Previous studies showed that Hsp27 and Hsp70 promote possessing aggresomes formed by a-synuclein and synphilin-1 are resistance to proteasome inhibitor-induced apoptosis (46, 47), and resistant to apoptosis (41), consistent with our conclusion that they they might also be expected to neutralize the cytotoxic effects of inhibit (rather than stimulate) cell death. It is tempting to protein aggregates independently of aggresome formation. We are speculate that the peripheral neuropathy observed in patients currently investigating the role that Hsps may play in resistance to treated with bortezomib (7) may be associated with aggresome bortezomib-induced apoptosis. formation and ER stress in peripheral neurons, which we plan to As noted above, other recent studies have implicated ER stress explore in future studies. Whether HDAC inhibitors exacerbate in the effects of bortezomib and other proteasome inhibitors in peripheral neuropathy will have to be watched closely in ongoing different tumor types (36, 37, 48). Our results confirm that cells clinical trials of the combination. undergoing apoptosis in response to bortezomib or bortezomib To further address the therapeutic potential of the combination plus SAHA displayed alterations consistent with ER stress, of bortezomib and HDAC inhibitors, we examined the antitumor including ER dilation, ER Ca2+ release, induction of ER stress- effects of bortezomib and SAHA in an L3.6pl orthotopic pancreatic associated genes (GADD34, CHOP, and Grp78/BiP), and Jun NH2- cancer model. Tumors exposed to the maximum tolerated dosage terminal kinase activation (Figs. 2 and 5; refs. 49, 50). However, we of bortezomib (1 mg/kg biweekly) displayed extensive aggresome believe that the strongest evidence for the involvement of ER stress formation (Fig. 6). Treatment with SAHA disrupted bortezomib- in bortezomib-induced cell death comes from mechanistic studies induced aggresome formation, decreased pancreatic tumor weight, that identified the caspases required for bortezomib-induced and enhanced tumor cell apoptosis (Fig. 6). We are currently apoptosis. Pioneering work with knockout mice established that ‘‘recycling’’ (24) the bortezomib-resistant Panc1 cells to enhance caspase-12 mediates ER stress-induced apoptosis in murine cells their tumorigenic potential so that the effects of combination (51), and bortezomib-induced apoptosis is also associated with therapy on an aggresome-positive, bortezomib-resistant tumor can caspase-12 activation in the mouse (28, 30, 37, 49, 50). However, be evaluated. Importantly, bortezomib did not induce aggresomes a recent study showed that expression of functional caspase-12 in in the normal human pancreatic epithelial cells in vitro nor in humans is limited to females of African descent (52), strongly normal murine pancreatic epithelial cells in vivo, and the normal suggesting that another caspase plays a more central role in ER cells did not undergo apoptosis in response to single-agent or stress-induced apoptosis in humans. The caspase-12 orthologue, combination therapy (Fig. 3; data not shown). The tumor cell caspase-4, is an excellent candidate, and a recent study confirmed selectivity of bortezomib or HDAC inhibitors has been reported that it is involved in ER stress-induced and amyloid-h-induced previously (42–44), and our data indicate that they also display apoptosis in human neuronal cells (53). Using immunofluorescence tumor selectivity when they are combined. In addition to the staining and confocal microscopy, we observed that caspase-4 immortalized normal pancreatic epithelial cells examined here, localizes to the ER in pancreatic cancer cells and is cleaved to its EBV-transformed normal B cells also failed to form aggresomes, active form in the four cell lines that undergo apoptosis in response whereas two multiple myeloma cell lines did so following to bortezomib (49). Furthermore, peptide-based or siRNA inhib- bortezomib exposure.8 itors of caspase-4 block bortezomib-induced apoptosis (49, 50). We do not have an explanation for why normal cells tend not to In ongoing work, we are investigating how bortezomib activates form aggresomes, but the question is currently under investigation. caspase-4 and how caspase-4 activation may initiate mitochondrial Increased cell proliferation is a hallmark of aggressive cancers events to promote cell death. and requires a general increase in protein synthesis and a heavy Taken together, our findings implicate ER stress in apoptosis dependency on proteasomal degradation of aged, misfolded, or induced by bortezomib in human pancreatic cancer cells (outlined oxidized proteins. Several of the signal transduction pathways (Ras, in Fig. 6C), and they provide the framework for clinical trials of phosphatidylinositol 3-kinase, mitogen-activated protein kinase, bortezomib and SAHA or other HDAC inhibitors in patients with and mammalian target of rapamycin) implicated in solid tumor pancreatic cancer. We have submitted a letter of intent to the Cancer progression activate various components of translation machinery Therapeutics Evaluation Program, NCI [H. Xiong, D.J. McConkey, (45). We speculate that the lower translation rates exhibited by and J.L. Abbruzzese (coprincipal investigators)] to do such a trial. normal cells make them resistant to bortezomib-induced aggresome formation and subsequent proteotoxicity. Although aggresomes seem to play important cytoprotective Acknowledgments functions in cells exposed to bortezomib, aggresome formation was Received 8/18/2005; revised 12/1/2005; accepted 1/26/2006. not the only mechanism of drug resistance we observed in our cell Grant support: American Legion Auxiliary, Sowell-Huggins families, and the Presidents of the University of Texas Health Science Center and M.D. Anderson Cancer lines. Two of them (MiaPaCa-2 and Hs766T) remained relatively Center (S.T. Nawrocki and J.S. Carew) and National Cancer Institute grants U54 CA resistant to bortezomib when it was combined with trichostatin A 090810 and P50 CA101936 (D.J. McConkey). The costs of publication of this article were defrayed in part by the payment of page or SAHA presumably because the cells did not form aggresomes in charges. This article must therefore be hereby marked advertisement in accordance response to proteasome inhibition. Consistent with our in vitro with 18 U.S.C. Section 1734 solely to indicate this fact.

8 S. Nawrocki et al., in preparation. 9 S. Nawrocki, unpublished data.

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