Cancer Therapy: Preclinical

Targeted Inhibition of Cyclic AMP -4 Promotes Brain Tumor Regression Patricia Goldhoff,1Nicole M. Warrington,1David D. Limbrick, Jr.,2 Andrew Hope,3 B. Mark Woerner,1 Erin Jackson,8 Arie Perry,4 David Piwnica-Worms,5,8 and Joshua B. Rubin1, 6,7

Abstract Purpose: As favorable outcomes from malignant brain tumors remain limited by poor survival and treatment-related toxicity, novel approaches to cure are essential. Previously, we identified the cyclic AMP phosphodiesterase-4 (PDE4) inhibitor as a potent antitumor agent. Here, we investigate the role of PDE4 in brain tumors and examine the utility of PDE4 as a thera- peutic target. Experimental Design: Immunohistochemistry was used to evaluate the expression pattern of a subfamily of PDE4, PDE4A, in multiple brain tumor types. To evaluate the effect of PDE4A on growth, a brain-specific isoform, PDE4A1was overexpressed in xenografts of Daoy medulloblas- toma and U87 glioblastoma cells.To determine therapeutic potential of PDE4 inhibition, Rolipram, temozolomide, and radiation were tested alone and in combination on mice bearing intracranial U87 xenografts. Results: We found that PDE4A is expressed in medulloblastoma, glioblastoma, oligodendro- glioma, ependymoma, and meningioma. Moreover, when PDE4A1was overexpressed in Daoy medulloblastoma and U87 glioblastoma cells, in vivo doubling times were significantly shorter for PDE4A1-overexpressing xenografts compared with controls. In long-term survival and bio- luminescence studies, Rolipram in combination with first-line therapy for malignant gliomas (temozolomide and conformal radiation therapy) enhanced the survival of mice bearing intra- cranial xenografts of U87 glioblastoma cells. Bioluminescence imaging indicated that whereas temozolomide and radiation therapy arrested intracranial tumor growth, the addition of Rolipram to this regimen resulted in tumor regression. Conclusions: This study shows that PDE4 is widely expressed in brain tumors and promotes their growth and that inhibition with Rolipram overcomes tumor resistance and mediates tumor regression.

Despite 30 years of clinical trials, favorable outcomes from breast overexpress , and a survey of 60 malignant brain tumors continue to be limited by poor survival different human tumor cell lines indicated that the majority of and excessive treatment-related toxicity (1). Targeted therapies this PDE activity was due to PDE4 (4). The PDE4 family of based on tumor biology hold enormous promise but have yet cAMP phosphodiesterases regulates cAMP levels through their to be proven clinically effective (2). Thus, it is essential to hydrolytic activity (5). There are four PDE4 subfamily continue our efforts to identify novel regulators of brain tumor (A-D), from which at least 20 different functional isoforms growth that can be targeted for effective therapies. are generated. These allow for tissue-specific intracellular Recent work from our laboratory suggested that cyclic AMP compartmentalization of cAMP signaling (5). Through distinct (cAMP) phosphodiesterase-4 (PDE4) might constitute a novel combinations of localization motifs, regulatory sites, and target for the treatment of brain tumors (3). Many human protein-protein interacting domains, this array of PDE4 tumor cells originating in the central nervous system, lung, and molecules performs numerous and often exquisitely specific

Authors’ Affiliations: Departments of 1Pediatrics, 2Neurosurgery, 3Radiation The costs of publication of this article were defrayed in part by the payment of page Oncology, 4Pathology and Immunology, 5Molecular Biology and Pharmacology, charges. This article must therefore be hereby marked advertisement in accordance 6Anatomy and Neurobiology, and 7Neurology and 8Molecular Imaging Center, with 18 U.S.C. Section 1734 solely to indicate this fact. Mallinckrodt Institute of Radiology,Washington University School of Medicine and Note: Supplementary data for this article are available at Clinical Cancer Research St. Louis Children’s Hospital, St. Louis, Missouri Online (http://clincancerres.aacrjournals.org/). Received 3/31/08; revised 7/16/08; accepted 8/6/08. P. Goldhoff, N.M. Warrington, and D.D. Limbrick, Jr., contributed equally to these Grant support: The Children’s Brain Tumor Foundation and The Pediatric Brain studies. Tumor Foundation (J.B. Rubin), Pilot Study Funding from the Molecular Imaging Requests for reprints: Joshua B. Rubin, Division of Pediatric Hematology/ Center at Washington University School of Medicine and the NIHP50 CA94056 Oncology, Department of Pediatrics, Washington University School of Medicine, (D. Piwnica-Worms), National Cancer Institute Cancer Center Support Grant P30 Campus Box 8208, 660 South Euclid Avenue, St. Louis, MO 63110. Phone: 314- CA91842 (High Speed Cell Sorter Core), NIH Neuroscience Blueprint Core Grant 286-2790; Fax: 314-286-2892; E-mail: [email protected]. P30 NS057105 to Washington University (Viral Vectors Core), and NIHR21CA F 2008 American Association for Cancer Research. 108 677 (MicroRT). doi:10.1158/1078-0432.CCR-08-0827

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that precise localization of phosphodiesterase action, such as Translational Relevance PDE4A1 to the region of the Golgi, is essential for the compartmentalization and appropriate transduction of cAMP- Therapies that can overcome the resistance of malignant dependent processes (18). The present studies establish a brain tumors would be a major clinical advance. Here, we previously unknown role for PDE4A1 as a positive regulator of investigate the role of cAMP PDE4 in stimulating brain intracranial brain tumor growth and thus implicate mediators tumor growth and the therapeutic utility of cAMP PDE4 in the region of the Golgi in this activity. inhibition in the treatment of malignant brain tumors. cAMP PDE4 was widely expressed in human brain tumors of glial The importance of PDE4 as a therapeutic target is evidenced and neuronal lineage, and forced expression of PDE4A1 by the effect of Rolipram on intracranial tumor growth and accelerated intracranial glioblastoma and medulloblastoma survival over a 5-month period when administered in combi- xenograft growth. Moreover, targeted inhibition of PDE4, in nation with temozolomide and conformal radiation therapy combination with standard radiation and chemotherapy, (a current first-line treatment for glioblastoma). In this regard, induced a unique regression of established intracranial glio- we show that PDE4 inhibition can promote regression of blastoma xenografts. These findings identify PDE4 as a malignant brain tumors when administered as an adjunct to novel molecular target for brain tumor therapy and indicate established therapies. that PDE4 inhibition should be evaluated in clinical trials for malignant brain tumors.

Materials and Methods

All animals were used in accordance with an established Animal functions (6). For instance, using interfering RNA, Lynch et al. Studies Protocol approved by the Washington University School of was able to show that only PDE4D5 regulated protein kinase A- Medicine Animal Studies Committee. dependent heterotrimeric G protein switching by h2-adrenergic receptors (7). Similarly, dominant-negative constructs allowed Chemicals and other reagents McCahill et al. to determine that PDE4D3 and PDE4C2, but not All chemicals were obtained from Sigma unless otherwise indicated. PDE4A4 or PDE4B1, were specifically required to regulate the All tissue culture reagents and media were obtained from Invitrogen basal activity of AKAP450-tethered protein kinase type II (8). unless otherwise indicated. A construct containing mCherry cDNA was the kind gift of Dr. Roger Y. Tsien (University of California). Murine Finally, targeted deletion of the PDE4 genes has also indicated PDE4A1 (accession no. AJ297396) was provided by Dr. James Cherry that PDE4 isoforms perform nonredundant functions such as (Boston University). The human PDE4A-specific antibody was - the specific requirement for PDE4B in lipopolysaccharide induc- rously provided by Dr. Miles Houslay (University of Glasgow). tion of tumor necrosis factor-a secretion in macrophages (9). The importance of PDE4 to tumor biology was first suggested Tumor cell lines by in vitro studies describing the antitumor activity of the The following experiments use the well-characterized U87 glioblas- specific PDE4 inhibitor Rolipram. Rolipram exhibited signifi- toma and Daoy medulloblastoma cell lines as models for human brain cant antigrowth effects when tested in vitro against several tumors. In previous in vitro and intracranial xenograft experiments with breast and lung carcinoma cell lines (10, 11), inhibiting growth these cell lines, we have characterized the role of CXCR4 and cAMP in the regulation of brain tumor growth and the antitumor effects of by up to 60% in mammary carcinoma and melanoma cells CXCR4 antagonists and cAMP-elevating drugs (3, 19). These studies (10). Furthermore, in vivo work in our laboratory showed that form the basis for the present work in which mCherry-expressing and Rolipram could slow the intracranial growth of glioblastoma mCherry-PDE4A1-expressing U87 and Daoy cells were generated via and medulloblastoma xenografts (3). lentiviral infection of previously generated enhanced green fluorescent Several studies have examined the mechanism of the protein-firefly luciferase-expressing cell lines as described (19–21). antitumor effects of Rolipram. We found that Rolipram inhibits Viral particles were produced by the Viral Vectors Core Facility of The growth by elevating intracellular cAMP levels (3), which was Hope Center for Neurological Diseases at Washington University consistent with the findings of McEwan et al. in studies of School of Medicine. Expression vectors used in viral production Rolipram treatment of colon carcinoma cells (12). Additional- contained either a transgene for mCherry fluorescent protein under a ly, Chen et al. determined that Rolipram inhibits proliferation CMV promoter alone or the CMV promoter-mCherry cassette together with a murine PDE4A1 (mPDE4A1) transgene under a ubiquitin in A172glioblastoma cells through the induction of the cell promoter. mCherry-expressing cells were sorted and collected by high- cycle inhibitors p27 and p21 (13). Despite these findings, speed fluorescence-activated cell sorting (MoFlo High-Performance Cell though, it remains unclear how cAMP elevation regulates Sorter; DAKO; Supplementary Fig. S1A). Cell lines are referred to as growth. U87-luc, mCherry-U87-luc, PDE4A1-U87-luc, mCherry-Daoy-luc, and In this study, we provide the first direct in vivo evidence that PDE4A1-Daoy-luc. PDE4 is a regulator of brain tumor growth and an important therapeutic target. We show that PDE4A expression is common Generation of intracranial xenografts in human brain tumors. Further, we show that the expression Intracranial xenografts were generated as described previously (3). level of a brain-specific isoform of PDE4, PDE4A1, is correlated Homozygous NCR nude mice (Taconic Farms) were anesthetized with the rate of intracranial xenograft growth. PDE4A1 is a [intraperitoneal ketamine (87 mg/kg)/xylazine (13 mg/kg); Phoenix Pharmaceuticals], the cranium was exposed, and a small hole was made unique ‘‘super-short’’ form of PDE4 (14). It possesses a 2mm lateral and posterior to the bregma with a size 34 inverted cone truncated amino terminus that lacks the regulatory and burr (Dremel). Mice were positioned in a stereotactic frame (Stoelting) protein-protein interacting domains that characterize the longer and 50,000 cells in 5 AL PBS were injected through a 27-gauge needle forms of PDE4 but which confers localization to the Golgi and over 1 min at 3 mm below the dura mater. The incision was closed with Golgi-derived vesicles (15–17). Huston et al. have suggested Vetbond (3M).

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Bioluminescence imaging mCherry-expressing intracranial xenograft tissue (mCherry-U87-luc, NCR nude mice bearing intracranial xenografts of U87-luc, mCherry- PDE4A1-U87-luc, mCherry-Daoy-luc, and PDE4A1-Daoy-luc) was U87-luc, PDE4A1-U87-luc, mCherry-Daoy-luc, or PDE4A1-Daoy-luc removed under direct fluorescence microscopy and frozen in liquid were injected with 150 Ag/g D-luciferin (Biosynth) as described (3). nitrogen. Frozen tissue was weighed and homogenized with 10 volumes  After anesthesia using 2.5% isoflurane, mice were imaged with a charge- of 10% ice-cold TCA and then centrifuged for 10 min at 1,500 g to coupled device camera-based bioluminescence imaging system (IVIS remove precipitate. The supernatant was washed three times with 50; Xenogen; exposure time 1-30 s, binning 8, field of view 12, f/stop 1, 8 volumes of water-saturated ether. The aqueous phase was dried down open filter). Signals were displayed as photons/s/cm2/sr (22). Regions and resuspended in cAMP assay buffer. Absorbance at 405 nm was of interest were defined manually and images were processed using measured, and cAMP concentrations were calculated based on a Living Image and IgorPro Software (Version 2.50) as described (3). Raw standard curve. Protein concentrations were measured by colorimetric data were expressed as total photon flux (photons/s; ref. 22). assay (Bio-Rad) according to the manufacturer’s directions. cAMP values were normalized to protein for each sample individually. Treatment of mice bearing intracranial xenografts Western blot analysis NCR nude mice bearing intracranial xenografts of U87-luc glioblas- toma cells were imaged at least twice after implantation of cells to Tissue culture extracts were prepared by lysing cells with radio- identify those with growing tumors. As described previously, 2weeks immunoprecipitation assay buffer [50 mmol/L Tris (pH 8.0), 150 after tumor cell implantation, cohorts of mice were divided into control mmol/L NaCl, 0.5% sodium deoxycholate, 0.1% SDS, and 1% Triton- and treatment groups (7-10 mice per group; refs. 3, 19). X-100] supplemented with inhibitor cocktail set II Systemic therapies. Rolipram and temozolomide (Pharmacy, St. (Calbiochem) and a protease inhibitor cocktail (Roche). Tumor lysates A Louis Children’s Hospital) treatments were administered via the were prepared by sonication in lysis buffer (100 L/mg tissue). The drinking water. Drug concentrations in the water were adjusted based on measured daily water consumption to deliver 5 mg/kg/d Rolipram  continuously or 21 mg/kg/d 5 days/mo temozolomide. Table 1. PDE4A expression in human brain tumors Irradiation. Mice were treated with conformal radiotherapy as described previously (23). Subject animals were anesthetized [intra- Patient Diagnosis Age (y) Sex PDE4A peritoneal ketamine (87 mg/kg)/xylazine (13 mg/kg)] and positioned c within a custom-head immobilization device affixed to the microRT 1 Medulloblastoma-c* 13 M 2 animal couch. The microRT couch was positioned within the microRT 2 Medulloblastoma-c 10 M 2 3 Medulloblastoma-d 8 F 1 device using a three-axis translational couch positioning system. To 4 Medulloblastoma-d 7M 1 create the radiotherapy plan, conformal radiotherapy fields were 5 Medulloblastoma-d 19 M 2 designed using the treatment planning system based on a computed 6 Medulloblastoma-d 2 F 2 tomography scan of the anesthetized and immobilized mouse using a 7Medulloblastoma-d 35 M 1 Phillips Brilliance CT scanner (Phillips Medical Systems). Treatment 8 Medulloblastoma-a 5 M 3 fields were hand-optimized to minimize exposure to the mucous 9 Astrocytoma-Ib 7M3 membranes of the oral cavity, oropharynx, nasal structures, esophagus, 10 Astrocytoma-I 8 F 1 trachea, eyes, and ears. Final plan consisted of two fields per fraction 11 Astrocytoma-I 12 F 3 using right and left lateral beams. For each fraction, each animal was 12 Astrocytoma-I 8 F 1 13 Astrocytoma-II 47M 3 treated per the radiotherapy plan using a clinical I-192HDR after- 14 Astrocytoma-II 57M 2 loading system (Nucletron) collimated via the microRT collimator 15 Astrocytoma-III 38 M 2 f system. Dose rate was 90 cGy/min. Each animal was treated with six 16 Astrocytoma-III 24 M 3 fractions of 5 Gy, prescribed to midplane, for a total of 30 Gy. Fractions 17Astrocytoma-III 47F 3 were delivered every other day (Monday, Wednesday, and Friday) for 2 18 Astrocytoma-III 21 F 1 consecutive weeks. Animals were observed to tolerate this regimen well. 19 Astrocytoma-III 73 F 1 20 Astrocytoma-IV 62 F 1 Immunohistochemistry 21 Astrocytoma-IV 50 F 3 In accordance with an institutional review board-approved protocol 22 Astrocytoma-IV 57F 2 for human research, human brain tumor tissue was retrieved from the 23 Astrocytoma-IV 60 F 4 24 Oligodendroglioma-II 44 F 1 pathology files at Washington University School of Medicine. Formalin- 25 Oligodendroglioma-II 50 F 1 fixed, paraffin-embedded tissue was processed and analyzed as 26 Oligodendroglioma-III 44 F 2 described previously (24). Murine-specific PDE4A antibody was applied 27Meningioma-I 65 F 1 at a concentration of 0.33 Ag/mL (Fabgennix), detected using biotin- 28 Meningioma-I 70 F 3 conjugated secondary antibodies augmented by streptavidin-horserad- 29 Meningioma-II 72 F 2 ish peroxidase, and visualized using 3,3¶-diaminobenzidine (DAKO). 30 Meningioma-III 29 F 3 Human-specific PDE4A was diluted 1:400 and detected as above. 31 Meningioma-III 70 F 3 Application of isotype-specific IgG to serial sections, in the absence of 32 Meningioma-III 61 M 0 PDE4A-directed antibody, served as controls for the specificity of 33 Ependymoma-II 18 M 1 34 Ependymoma-II 68 F 2 antibody staining. 35 Ependymoma-III 7M 2 36 Ependymoma-III 39 M 0 cAMP measurement 37Ependymoma-III 6 F 2 cAMP was measured by competitive immunoassay using a Correlated Immunoassay Kit (Assay Designs) according to the manufac- * turer’s instructions (3). Briefly, mCherry-U87-luc, PDE4A1-U87-luc, Histologic subtypes of medulloblastoma: c, classic; d, desmo- plastic; a, anaplastic. mCherry-Daoy-luc, and PDE4A1-Daoy-luc cells growing in fetal bovine c a PDE4A expression grading: 1, 1-25%; 2, 26-50%; 3, 51-75%; 4, serum-supplemented (10%) -MEM (Life Technologies) were lysed in 76-100% of tumor cells are positive for PDE4A expression. 0.1 mol/L HCl and particulate matter was removed by centrifugation bHistologic grading of brain tumors based on the WHO classifica- at >600  g for 10 min. The supernatants were dried down and tion, grades I to IV. resuspended in cAMP assay buffer (Assay Designs). Alternatively,

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analyzed using the generalized estimating equation regression analysis. For cAMP comparisons, significance was determined by two-tailed t test. Statistical significance was assumed for P < 0.05.

Results

cAMP PDE4 is widely expressed in human brain tumors. The antitumor effect of Rolipram suggested that PDE4 was important in brain tumor biology. Because members of the PDE4A subfamily are distributed throughout the brain (25), we examined the expression pattern of PDE4A in 37 tumors from pediatric and adult patients (Table 1). Among these were four types of parenchymal brain tumors (astrocytomas, medullo- blastomas, oligodendrogliomas, and ependymomas) as well as meningiomas (nonparenchymal, intracranial tumors) of vari- ous grades and histologic subtypes. PDE4A was expressed in all tumor types examined (Supplementary Fig. S2). Staining in tumor cells was most commonly punctate and frequently perinuclear (Fig. 1). All astrocytomas (15) and medulloblasto- Fig. 1. PDE4A is expressed in a punctate pattern in brain tumors. Representative mas (8) exhibited PDE4A expression with no correlation section of an ependymoma stained for PDE4A (brown). Bar, 10 Am. between grade or histologic subtype, respectively. In more limited sample sizes, the majority of ependymomas (4 of 5) proteins were resolved with 4% to 12% or 10% Bis-Tris gels (Invitrogen) and transferred onto Hybond ECL nitrocellulose mem- and meningiomas (5 of 6) and all oligodendrogliomas (3 of 3) brane (Amersham) according to standard protocols. Membranes were showed PDE4A expression. In most cases, PDE4A expression incubated with antibodies directed against murine PDE4A (1:1,000; was found solely in tumor cells. In several specimens, however, Abcam), human PDE4A (1:400; Houslay), and actin (1:3,000; Sigma) PDE4A was also evident in endothelial cells. Leukocytes within overnight at 4jC. This was followed by incubation with horseradish the lumens of tumor-associated blood vessels were consistently peroxidase-conjugated secondary antibody (1:20,000; Bio-Rad). Perox- stained (data not shown). Thus, PDE4A expression is wide- idase activity was detected using the enhanced chemiluminescence spread in brain tumors. SuperSignal West Pico System (Pierce). Quantitation of Western blots cAMP PDE4A1 stimulates brain tumor growth in vivo. To was done by densitometry using ImageJ software from the NIH. determine whether the high level of PDE4A expression in Statistical analysis human brain tumors was indicative of a growth advantage, we Data were analyzed using GraphPad Prism version 4.00 (GraphPad examined the effect of increased PDE4A expression on brain Software) or Stata10 (Stata). Kaplan-Meier survival curves were tumor growth. We overexpressed a murine homologue of a analyzed using pairwise log-rank tests. Given the repeated measure- brain-specific isoform of PDE4, mPDE4A1 (26), via lentiviral ments of mice over time, statistical differences in growth curves were infection of U87 glioblastoma and Daoy medulloblastoma

Fig. 2. Overexpression of PDE4A1stimulates intracranial tumor growth. mCherry-Daoy-luc, or mCherry-U87-luc, or PDE4A1-Daoy-luc, or PDE4A1-U87-luc cells were stereotactically implanted into the cortex of nude mice. A, tumor growth was measured by weekly bioluminescence imaging. Bioluminescence ratios (total photon flux each week/photon flux from week 1). P values for differences between control and PDE4A1-overexpressing curves were determined by generalized estimating equation regression analysis. B, human and murine PDE4A expression was evaluated by Western blot analysis of lysates derived from isolated xenograft tumor tissue. Asterisk, human PDE4A1. C, cAMP levels were determined by ELISA from diethyl ether extracts of control and PDE4A1-overexpressing Daoy and U87 mCherry-positive tumor tissue isolated under direct fluorescence microscopy. Mean and SE values normalized to mCherry controls for both U87 and Daoy tumor tissue samples (n =13). **, P < 0.01, two-tailed t test.

Clin Cancer Res 2008;14(23) December 1,2008 7720 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2008 American Association for Cancer Research. PDE4 Inhibition Promotes BrainTumor Regression cells that have been engineered previously to express enhanced luminescence imaging weekly and were examined for signs of green fluorescent protein and firefly luciferase (Supplementary advancing disease such as weakness, weight loss, and seizures. Fig. S1A). As expected, increased mPDE4A1 expression (Sup- Mice observed to be suffering from their disease were plementary Fig. S1B) was correlated with lower levels of cAMP euthanized. compared with mCherry control cells (Supplementary Fig. S1C). All treatments were associated with improved survival To better understand how PDE4A1 might regulate tumor compared with controls (Fig. 3). Pairwise log-rank tests growth in vivo, mCherry-expressing (control) or mPDE4A1- indicated significant differences in survival between control overexpressing brain tumor cells were stereotactically (no treatment) and the following regimens: temozolomide implanted into the brains of nude mice and intracranial (P = 0.0016); radiation (P = 0.0005); radiation and temozo- growth was measured by weekly bioluminescence imaging as lomide (P < 0.0001); Rolipram and temozolomide (P < described previously (3). In both U87 and Daoy cell xenografts, 0.0001); Rolipram and radiation (P = 0.0006); and radiation, overexpression of mPDE4A1 was associated with significantly temozolomide, and Rolipram (P < 0.0001). In terms of overall accelerated intracranial growth (Fig. 2A). Curve fitting of the survival, however, the treatments could be divided into two relation between bioluminescence ratios and time [exponential groups: minimally effective (Fig. 3A) and highly effective growth: Y = start  exp(k  X), where k = rate constant and therapies (Fig. 3B). The minimally effective therapies included tumor doubling time = 0.69 / k] revealed that expression of temozolomide, radiation therapy, Rolipram, and Rolipram and mPDE4A1 in Daoy cells was associated with a decrease in radiation therapy; these mice had an overall survival of V42%. doubling time from 1.68 to 1.09 weeks. Similarly, over- The highly effective therapies included Rolipram and temozo- expression of mPDE4A1 in U87 cells produced a decrease in lomide, radiation therapy and temozolomide, and Rolipram, tumor doubling time from 0.94 to 0.39 weeks. radiation therapy, and temozolomide. Survival increased To evaluate the relationship between PDE4A1 expression significantly among mice receiving one of these three therapies. and intracranial tumor growth, mCherry-positive tumor tissue Interestingly, the combination of Rolipram and temozolomide was recovered from control and mPDE4A1-overexpressing (70% survival) was similar in activity to radiation and xenografts under direct fluorescence microscopy (Supplemen- temozolomide (a current first-line therapy; 70% survival), tary Fig. S3). Western blot analysis of mCherry-U87-Luc and whereas the addition of Rolipram to radiation therapy and mCherry-Daoy-Luc tumor tissue with the human-specific temozolomide provided the greatest effect on survival with only PDE4A antibody revealed multiple human PDE4A (hPDE4A) a single death over the 150-day experiment (90% survival). isoforms including one with an apparent molecular weight of Bioluminescence imaging distinguishes treatment effects 78 kDa consistent with hPDE4A1 (ref. 14; Fig. 2B). Western blot in vivo. To gain insight into how these treatments improved analysis of mCherry control and mPDE4A1-overexpressing survival, we examined the bioluminescence imaging data for Daoy and U87 xenografts with the murine-specific PDE4A antibody confirmed continued xenograft expression of mPDE4A1. Consistent with in vitro studies (Supplementary Fig. S1), overexpression of mPDE4A1 in vivo was evident as a prominent band at 68 kDa (Fig. 2B). In addition, a second band of 83 kDa was evident. This latter band is similar to one pre- viously reported for mPDE4A1 expression in COS-7 cells (27). To verify the functionality of the overexpressed PDE4A1, cAMP levels were examined in mCherry-positive tumor tissue. cAMP was extracted from xenograft tumor tissue and measured by ELISA as described previously (3). Increased mPDE4A1 expression was associated with decreased levels of cAMP in the tumor tissue (Fig. 2C). These data are consistent with a role for PDE4 in the stimulation of intracranial brain tumor growth. Rolipram enhances the survival of mice bearing intracranial xenografts. The growth-promoting activity of PDE4A1 suggests that targeted inhibition of PDE4 with the specific inhibitor Rolipram could be an important addition to current brain tumor therapies. We therefore performed long-term survival and coupled bioluminescence studies in mice bearing intra- cranial xenografts of U87-luc glioblastoma cells to determine the effects of Rolipram treatment in combination with standard chemoradiation. Therapy began 2weeks after tumor cell implantation. This time was sufficient to establish steadily increasing biolumines- cence (a measure of tumor growth) in the xenografts. Mice were Fig. 3. Inhibition of PDE4 with Rolipram enhances survival in mice bearing assigned to one of eight treatment groups: control, Rolipram, intracranial U87 xenografts. Nude mice bearing intracranial U87-luc xenografts temozolomide, radiation therapy, Rolipram and radiation were assigned to eight treatment groups (7-10 mice per group) and left therapy, Rolipram and temozolomide, radiation therapy and untreated (control) or treated with Rolipram, temozolomide, radiation therapy, or combinations thereof as described in Materials and Methods. Survival was temozolomide (a current first-line therapy), and Rolipram, monitored over 150 days. Data are presented as a Kaplan-Meier curve for radiation therapy, and temozolomide. Mice underwent bio- (A) minimally effective treatments and (B) highly effective treatments.

www.aacrjournals.org 7721 Clin Cancer Res 2008;14(23) December 1, 2008 Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2008 American Association for Cancer Research. Cancer Therapy: Preclinical evidence of antitumor growth effects. All mice, whether alive or Tumors in temozolomide-treated mice showed a multiphasic dead, were included in the analysis. For those mice that had growth response with an initial regression timed to the died, their last recorded total photon flux was included in completion of the second course of therapy (Fig. 4C). generating the mean photon flux for the group. Biolumine- Exponential growth resumed after 8 weeks, however, with scence data for evaluating treatment efficacy was analyzed by the midpoint at 12.3 weeks and a maximal slope of 8.9 Â determining the midpoint of maximal growth and rate of 109 photons/wk. maximal growth in control (untreated) mice and comparing it In contrast to the minimally effective therapies (Fig. 4), the with the other treatment groups. highly effective therapies produced more favorable results on Several informative patterns of intracranial growth emerged. intracranial growth. With each of these treatments, no tumor Tumors in control mice grew in an uninterrupted, exponen- growth beyond an initial phase of 2to 3 weeks was observed tial fashion and exhibited a midpoint of maximal growth at (Fig. 5). Consistent with the survival data, radiation and 6.5 weeks and maximal exponential growth of 7.8 Â temozolomide (Fig. 5A) versus Rolipram and temozolomide 109 photons/wk (Fig. 4). Because all control mice died by (Fig. 5B) were indistinguishable with regard to their inhibition week 10, the plateau represents the mean of the final measures of intracranial growth. Of note, bioluminescence signal of all mice in this group. persisted in mice treated with either of these regimens, Interestingly, although radiation and Rolipram had similar indicating that although these tumors were not growing, they effects on overall survival, the two treatments had markedly remained viable. The addition of Rolipram to radiation and different effects on growth. Compared with control, Rolipram temozolomide resulted in tumor regression (Fig. 5C and D). treatment did not appreciably alter the midpoint of maximal After 12weeks of this therapy, bioluminescence declined to growth, which was 6.7 weeks in the latter group (Fig. 4A); f1% of the starting values. Together, these data indicate that however, Rolipram alone decreased the rate of maximal tumor Rolipram provides a unique therapeutic effect that promotes growth to 1.8 Â 109 photons/wk. tumor regression and enhances survival. In contrast, radiation therapy induced a transient arrest in growth, delaying the midpoint of maximal growth to 10.5 Discussion weeks (Fig. 4B). However, after recovery from radiation, the rate of exponential growth in irradiated tumors was similar to that In this study, we show that PDE4A1 overexpression of control mice, with a slope of 5.7 Â 109 photons/wk. The stimulates brain tumor growth in vivo, whereas inhibition of combination of radiation and Rolipram exhibited similar PDE4 suppresses tumor growth and augments the antitumor antitumor effects as radiation alone (Fig. 4D). effects of chemotherapy and radiation therapy. PDE4 has been

Fig. 4. Bioluminescence imaging reveals modes of antitumor effects. Intracranial tumor growth was measured by serial bioluminescence imaging. Mean photon flux over time for the minimally effective therapies (see Fig. 3) compared with control. A, Rolipram. B, radiation. C, temozolomide. D, radiation and Rolipram.

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Fig. 5. Addition of Rolipram to standard radiation therapy and temozolomide results in tumor regression. Intracranial tumor growth was measured by serial bioluminescence imaging. Mean photon flux over time for the highly effective treatments (see Fig. 3) compared with control. A, radiation and temozolomide. B, Rolipram and temozolomide. C, Rolipram, radiation, and temozolomide. D, comparison of Rolipram, radiation, and temozolomide to the current standard of care, radiation, and temozolomide.

shown previously to be widely expressed in several different 32–34). The current study lends additional support to this human tumor cell lines (4). In addition, we now show that model as the effects of PDE4A1 overexpression and Rolipram PDE4A is also expressed in astrocytomas, medulloblastomas, are most likely the result of changes in cAMP levels. The work of oligodendrogliomas, ependymomas, and meningiomas, indi- Houslay and Adams has shown that PDE4s play multiple roles cating that PDE4A activity may be essential to the growth of all in intracellular signaling. These roles include the regulation of brain tumors. receptor desensitization and G protein switching as well as To better define the role of PDE4 in brain tumor biology, we protein kinase A and extracellular signal-regulated kinase overexpressed PDE4A1, a brain-specific isoform, in U87 activation (5). There are at least 20 different isoforms of glioblastoma and Daoy medulloblastoma cells (26). Stimula- PDE4 that can be inhibited with Rolipram. Each contains a tion of growth occurred in both brain tumor models. U87 cells unique NH2-terminal region that appears to regulate specific were derived from a malignant astrocytoma. These tumors compartmentalized functions (6). For example, the NH2- typically carry mutations in the mitogen-activated protein terminal regions of PDE4A4 and PDE4A5 interact with the kinase, phosphatidylinositol 3-kinase, Rb, and MDM2/p53 SH3 homology domains of Src family kinases, whereas the pathways (28), and consistent with this, U87 cells possess NH2-terminal region of PDE4A1 facilitates membrane inser- deletion of p14/p16 and PTEN genes (29). Daoy cells were tion, particularly into the trans-Golgi membrane (35). Thus, derived from a desmoplastic medulloblastoma. Medulloblas- overexpression of even catalytically inactive PDE4 isoforms can tomas are characterized by mutational activation of the sonic alter signaling functions by displacing active endogenous PDE4 hedgehog, Wnt, and Myc pathways (30). Daoy cells also carry a from necessary binding sites and functioning as dominant- mutation of TP53 (31). Despite these differences in molecular negative constructs (8). The PDE4A1 isoform lacks the profile, PDE4A1 overexpression stimulated both U87 and Daoy regulatory and protein-protein interacting domains that are growth in vivo. These data suggest that PDE4A1 effects were not found in longer PDE4 isoforms (5, 35). Thus, the growth- dependent on the locus into which the transgene inserted and promoting effects of PDE4A1 overexpression are most readily were not specific to pathways activated by mutation in these attributed to increased cAMP hydrolysis as evidenced by the tumors. Instead, these results indicate that PDE4A1 is a positive observed decrease in levels of cAMP. regulator of brain tumor growth. In this regard, the localization of PDE4A1 to the membranes It will be important to determine whether the cAMP- of the Golgi apparatus and Golgi-derived vesicles is of interest. hydrolyzing activity of PDE4A1 is essential to its growth- Although global changes in cAMP were detected in our cells promoting effects. Previously, we and others have suggested and xenografts overexpressing PDE4A1, PDE4 effects can also that low levels of cAMP stimulate brain tumor growth (3, 12, be limited to regulating local cAMP-dependent functions. For

www.aacrjournals.org 7723 Clin Cancer Res 2008;14(23) December 1, 2008 Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2008 American Association for Cancer Research. Cancer Therapy: Preclinical instance, targeted deletion of PDE4B revealed that it, and not mechanism by which Rolipram augments the activity of radia- PDE4A or PDE4D, was necessary for regulating TLR signaling in tion and temozolomide may be distinct from modulating macrophages (9). The effect of PDE4B loss on macrophage DNA alkylation. responses to lipopolysaccharide stimulation was dependent on The present study strongly indicates that PDE4 inhibitors protein kinase A activation but was not associated with global should be evaluated in clinical trials for brain tumors. Our data changes in cAMP levels. These data suggest that localization of suggest that PDE4 inhibition may be the key adjunct to PDE4B is necessary to regulate cAMP levels in a critical standard chemotherapy and radiation therapy to promote brain subcompartment for TLR signaling. Thus, it will be essential tumor regression. Furthermore, the equivalence of combined to identify Golgi-localized mediators of PDE4A1 effects on radiation and temozolomide to Rolipram and temozolomide tumor growth and to determine whether elevation of cAMP suggests that PDE inhibitors might prove to be suitable levels antagonizes their growth-promoting functions. alternatives for radiation when combined with chemotherapy. Elevation of cAMP as a therapeutic strategy for cancer has This may be especially important for young patients for whom been examined previously but was limited by the excessive irradiation is not an option due to the detrimental effects toxicity of the early pleiotropic phosphodiesterase inhibitors radiation has on their developing brains. (36, 37). Specific phosphodiesterase inhibitors such as Roli- The potential for Rolipram to be a useful agent in brain pram are better tolerated at therapeutic doses (38) and exhibit tumor therapy is emphasized by its ability to penetrate into and strong antitumor effects (10–12). Prior studies suggested a treat diseases of the central nervous system. However, clinical therapeutic role for Rolipram in the treatment of brain tumors application of Rolipram might also be limited by its toxicities. and colon cancer (3, 12). To our knowledge, this is the first In multiple clinical trials for depression and multiple sclerosis, report to show a survival advantage in response to Rolipram Rolipram was relatively well tolerated, with nausea and treatment over an extended preclinical trial. vomiting the most commonly reported side effect (38). The potential importance of Rolipram therapy was most However, in long-term toxicity studies in rats, high-dose evident on analysis of bioluminescence data pertaining to the Rolipram therapy was also associated with prolactinomas and highly effective therapies of radiation and temozolomide, mammary adenocarcinoma (41). Although serious, these side Rolipram and temozolomide, and Rolipram, radiation, and effects are not substantially different from the nausea, vomiting, temozolomide. Although the initial pattern of tumor growth and secondary cancer risks of commonly used treatments for was similar among these three groups, the value of Rolipram brain tumors like radiation therapy and temozolomide. treatment became most evident at 12weeks of therapy. Therapy Therefore, if Rolipram showed a unique therapeutic effect, its with either Rolipram and temozolomide or radiation and known toxicities would not preclude its use in the treatment of temozolomide resulted in cessation of further tumor growth, malignant brain tumors. However, newer PDE4 subfamily- but tumors remained viable as evidenced by their persistent specific inhibitors may prove equal or more efficacious and less bioluminescent signal. In contrast, treatment with Rolipram in toxic (6, 42). Thus, additional investigation into whether other conjunction with radiation and temozolomide resulted in PDE4 isoforms can also regulate brain tumor growth and nearly complete loss of bioluminescence, indicating tumor whether subfamily-specific inhibitors exhibit equal efficacy to regression. Rolipram is essential. Human studies have shown that a major determinant of response to alkylator therapy like temozolomide is the expres- Disclosure of Potential Conflicts of Interest sion and activity of the DNA repair enzyme methyl glutamyl methyltransferase (39). In patients with reduced methyl gluta- No potential conflicts of interest were disclosed. myl methyltransferase expression through promoter methy- lation, there is a greater response to temozolomide therapy. Acknowledgments Thus, one possible mechanism whereby Rolipram enhances the effects of temozolomide and radiation might be the inhibition We thank Erich Kiehl for assistance with mice irradiation, Michael Geske for help of methyl glutamyl methyltransferase. Consistent with pub- with statistical analysis, Mahil Rao for designing lentiviral constructs, Kristin Brown for editorial assistance, Drs. Jonathan Gitlin and Louis Muglia for critical reading of lished studies, we found that Daoy and U87 cells express the article, and the Alvin J. Siteman Cancer Center at Washington University methyl glutamyl methyltransferase (40), but this was unaffected School of Medicine and Barnes-Jewish Hospital for the use of the High Speed by Rolipram treatment in vitro (data not shown). Thus, the Cell Sorter Core.

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