Imiquimod Treatment Induces Expression of Opioid Growth Factor Receptor: a Novel Tumor Antigen Induced by Interferon-␣?

Vol. 10, 4959–4970, August 1, 2004 Clinical Cancer Research 4959

Featured Article Imiquimod Treatment Induces Expression of Opioid Growth Factor Receptor: A Novel Tumor Antigen Induced by Interferon-␣?

Mirjana Urosevic,1 Patrick A. Oberholzer,1 half of the cases. Expression of opioid growth factor receptor Tanja Maier,1 Ju¨rg Hafner,1 Elisabeth Laine,1 correlated with a longer recurrence-free period in basal cell 2 3 carcinoma that recurred after radiotherapy (Kaplan-Meier ؍ ,Herbert Slade, Bernd Benninghoff 1 1 analysis, P 0.041). Gu¨nter Burg, and Reinhard Dummer Conclusions: In addition to its immunomodulatory and 1Department of Dermatology, University Hospital Zurich, Zurich, 2 3 antiproliferative activity, opioid growth factor receptor Switzerland; 3M Pharmaceuticals, St. Paul, Minnesota; and 3M seems to have a prognostic significance in basal cell carci- Pharmaceuticals, Neuss, Germany noma patients. Our data add to the growing list of basal cell carcinoma-associated tumor antigens. ABSTRACT Purpose: Imiquimod represents a synthetic local im- INTRODUCTION mune response modifier that has demonstrated efficacy in Basal cell carcinoma is the most common malignancy in clearing basal cell carcinoma. Via interaction with Toll-like the white human population worldwide, and its incidence has receptor 7 on immune cells, imiquimod induces local pro- been increasing in the last decade (1). Basal cell carcinoma duction of cytokines, such as interferon (IFN)-␣. tends to grow slowly and rarely metastasizes. Basal cell carci- Experimental Design: To more closely define and eluci- noma can be successfully treated by intralesional injections of date mechanisms leading to basal cell carcinoma clearance interferons (IFNs) or by topical application of imiquimod (1). in vivo, we examined gene expression profiles of skin basal Imiquimod has been tested and found to be efficacious in cell carcinoma before and after treatment with 5% imi- clearing basal cell carcinoma [basal cell carcinoma (2–4)]. quimod cream (Aldara) by using high-density oligonucleo- Imiquimod [1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4- tide arrays. amine] belongs to the group of imidazoquinolones, synthetic Results: We show that imiquimod predominantly in- local immune response modifiers that have demonstrated potent duces genes involved in different aspects of immune re- antiviral and antitumor activity (5). Imiquimod activates macro- sponse. In addition to effects on immunity, imiquimod treat- phages and other monocyte-derived immune cells via interac- ment modulates the expression of genes involved in the tion with Toll-like receptor-7 (6) inducing the local production control of apoptosis and oncogenesis. Array data indicated of cytokines such as IFN-␣, tumor necrosis factor ␣, and inter- that imiquimod treatment induces expression of opioid leukin (IL)-12, resulting in an enhanced innate immune response growth factor receptor, a molecule recently reported to be a (5, 7). In addition, imiquimod induces migration and activation target for antitumor antibody responses. Immunohisto- of skin Langerhans cells (8), all of which promote the biasing of chemistry revealed in vivo up-regulation of opioid growth naı¨ve T cells to Th1 cells, which are potent producers of IFN-␥, factor receptor protein on tumor and on infiltrating cells thus leading to the enhancement of adaptive immunity. The after treatment. By using basal cell carcinoma cell lines stimulation of innate and adaptive immunity may finally lead to treated with IFN-␣ or imiquimod, we show that opioid regression of viral-induced lesions and neoplasms (5, 7). growth factor receptor up-regulation is IFN-␣-mediated, A functional genomic approach offers a unique possibility rather then directly imiquimod-mediated. By using tissue to define and analyze gene expression profiles in response to a microarray containing 52 basal cell carcinomas, we demon- specific drug treatment. The implementation of high-density strate opioid growth factor receptor expression in almost oligonucleotide arrays containing more than 10,000 genes on a single array has enabled such a genome-wide assessment of changes in the overall expression profile (9). Using this approach, we have analyzed in vivo gene expression profiles of Received 2/1/04; revised 4/27/04; accepted 5/7/04. basal cell carcinomas treated with 5% imiquimod cream. In Grant support: 3M Pharmaceuticals, Gottfried and Julia Bangerter- addition, we established basal cell carcinoma cell lines from Rhyner Foundation, and Functional Genomics Center Zurich. skin lesions, which we treated with imiquimod or IFN-␣, and The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked their gene expression profiles were then compared with the ones advertisement in accordance with 18 U.S.C. Section 1734 solely to from skin basal cell carcinoma after imiquimod therapy in vivo. indicate this fact. Note: Supplementary data for this article are available at Clinical Cancer Research online (http://clincancerres.aacrjournals.org). MATERIALS AND METHODS Requests for reprints: Reinhard Dummer, Department of Dermatol- Patient Samples ogy, University Hospital Zurich, Gloriastrasse 31, 8091 Zurich, Switzerland. Phone: 41-1-255-25-07; Fax: 41-1-255-89-88; E-mail: This substudy used material from a 3M Pharmaceuticals- [email protected]. supported study (10) investigating infiltrate alterations in skin

basal cell carcinoma after treatment with 5% imiquimod cream Diagnostics, Farmingdale, NY) and purified using RNeasy col- (Aldara) approved by Institutional Ethnics Committee. Before umns (Qiagen AG, Basel, Switzerland). Fifteen ␮g of biotiny- entering the study, all patients provided written informed con- lated cRNA were fragmented and hybridized to HG-U95Av2 sent for both studies. At the screening visit, a biopsy specimen GeneChip arrays (Affymetrix, Santa Clara, CA) that contain was taken for histological confirmation of skin basal cell carci- probe sets representing ϳ12,000 genes. Chip hybridization, noma. Patients applied the cream once daily 5 times/week for a washing, and staining were performed according to the maximum of 6 weeks. At the point when the tumor began to Affymetrix-recommended protocols. show signs of erosion, the tumor was surgically excised. The After scanning of the probe arrays, digitalized image data surplus material was available from five patients, one of whom were processed using Affymetrix Microarray Suite 5.0 software. had a second basal cell carcinoma lesion, which was treated on In addition to determining the expression level for each gene signed informed consent after completion of the study. An (the so-called “signal”), this software assigns a detection call additional patient with M. Bowen was treated according to the [absent (not expressed), present (expressed), or marginal (mar- same scheme, on signed informed consent. ginally expressed)] for each gene measured. The overall fluo- rescence intensity was scaled to a global intensity of 500 to enable the comparison between chips. Cell Lines Establishment of Primary Basal Cell Carcinoma Cell Data Analysis Cultures. Four primary basal cell carcinoma cell cultures were established according to the previously described method- The expression data for all genes were exported to Mi- ology (11). These basal cell carcinoma cell lines exhibited a low crosoft Excel. The following criteria were used to select differ- ␣ culture-doubling rate, ranging from 14 to 30 days. Basal cell entially expressed genes on imiquimod (or IFN- ) treatment: (a) Ͻ carcinoma cell lines expressed keratin markers detected by two paired Student’s t test significance level of P 0.05; (b) fold antibodies: Lu-5 (BMA Biomedicals AG, Augst, Switzerland) change cutoff of 1.5 in both directions; and (c) the present call(s) and NCL-L-AE1/AE3 (Medite AG, Nunningen, Switzerland). had to coincide with the regulation in question, e.g., if a candi- They were negative for expression of vimentin, neuron-specific date gene were to be called “up-regulated” after immunomodu- enolase, and S-100. To remove contaminating keratinocytes, the latory treatment, at least one sample in the treated group had to primary basal cell carcinoma cultures were exposed to “calcium be called present; vice versa, if a gene were to be called shift” (11). The increase in calcium concentration in growth “down-regulated” after immunomodulatory treatment, at least medium led to their cornification in the next 96 h, whereas basal one sample in the untreated group had to have a present call. The cell carcinoma cells flattened, elongated, and stratified but nei- selected differentially expressed genes were normalized to a ther cornified nor died (11). mean of 0 and a SD of 1, log2-transformed, and subjected to Immunomodulatory Treatment. The HCl salt form of average linkage hierarchical clustering using the uncentered imiquimod was provided by 3M Pharmaceuticals (St. Paul, Pearson similarity matrix (13). Clustering analysis was performed using the GENE CLUSTER program, and the figures MN). Primary basal cell carcinoma cell lines and human kera- 4 tinocytes were stimulated with 15 ␮g/ml imiquimod or with were generated with the TREE VIEW program (13). The 1000 units/ml IFN-␣2a (Roferon-A; Roche) for 24 h. Primary selected genes were annotated using the NetAffx analysis sys- human keratinocytes were obtained from foreskins as described tem offered by Affymetrix (14). elsewhere (12) and grown in defined keratinocyte serum-free medium (Gibco; Invitrogen AG, Basel, Switzerland) containing Real-Time Polymerase Chain Reaction bovine pituitary extract and recombinant epidermal growth Approximately 1 ␮g of total RNA was reverse transcribed factor. using oligo-p(dT)15 priming and avian myeloblastosis virus To evaluate the viability of the cells after the aforemen- reverse transcriptase [1st Strand cDNA Synthesis Kit for Re- tioned treatments, the cells were stained with propidium iodine verse Transcription-PCR (avian myeloblastosis virus); Roche (Fluka Chemie GmbH, Buchs, Switzerland) and counted under Molecular Biochemicals, GmbH, Mannheim, Germany] at 42°C an inverted epifluorescence microscope. Neither imiquimod nor for 1 h. PCR amplifications were carried out using HotStart IFN-␣ treatment induced a significant decrease in cell numbers system (LightCycler-Faststart DNA Master SYBR Green I; after 24 h (37.25 Ϯ 8.18 cells/cm2 before treatment versus Roche Molecular Biochemicals) in the LightCycler thermocy- 40.50 Ϯ 11.09 cells/cm2 after imiquimod treatment or 38.75 Ϯ cler, as described previously (15). The primer sets used and 14.5 cells/cm2 after IFN-␣ treatment). cycling conditions are shown in Supplementary Table 1. To generate external standards for subsequent quantification, PCR products were purified and cloned into the pCRII-TOPO vector Gene Expression Profiling using TOPO TA Cloning Kit (Invitrogen AG), according to the Total RNA was isolated using TRIzol reagent (Invitrogen manufacturer’s recommendations. The incorporation of the in- AG). Double-stranded cDNA was generated using a Superscript sert into the vector was confirmed by sequencing and BLAST cDNA synthesis kit (Invitrogen AG) using an oligo(dT)24 comparison. primer containing a T7 RNA polymerase promoter at the 3Ј end (Microsynth, Balgach, Switzerland). Labeled cRNA was pre- pared from double-stranded cDNA by in vitro transcription using a T7 polymerase [MEGAscript T7 kit; Ambion (Europe) Ltd.] in the presence of biotin-11-CTP and biotin-16-UTP (Enzo 4 http://rana.lbl.gov.

Table 1 OGFR protein expression in basal cell carcinomas treated Joseph A. Mollick (Department of Medicine, Division of On- with topical imiquimod cology, Stanford University Hospital, Stanford, CA). Immuno- Patient OGFR TU OGFR INF histochemistry was performed with a 1:30 working antibody P1 before ϩϩdilution using the alkaline phosphatase-anti-alkaline phospha- P1 after 5 days ϩϩϩ ϩϩϩ tase technique, as described previously (10). P2 before Ϯϩ P2 after 3 days ϩϩϩ ϩϩϩ P3 before ϮϪRESULTS P3 after 3 days ϩϩϩ ϩϩϩ P4 before ϪϮ We generated gene expression profiles for six basal cell P4 after 4 days ϩϩcarcinomas and one M. Bowen sample before and after treat- P5-1 before ϩϪment with 5% imiquimod cream and for four basal cell carci- P5-1 after 3 days ϩϩ ϩϩϩ ϮϪnoma cell lines and two keratinocyte controls before and after P5-2 before ␣ P5-2 after 3 days ϩϩ ϩϩϩ treatment with IFN- or imiquimod. Based on our filtering Px before ϪϪcriteria, we found the expression of 1305 genes (ϳ10% of the Px after 18 days ϩϩ ϩϩϩ genes on the HG-U95Av2 chip) to be differentially regulated on MB before ϩϩtreatment with imiquimod of the skin basal cell carcinoma. Of ϩϩϩ ϩϩϩ MB after 5 days these genes, 372 (28.5%) genes were up-regulated, whereas 933 NOTE. The duration of the treatment is presented in days (time (71.5%) genes were down-regulated. Two-way clustering anal- point where the tumor showed signs of erosion and was excised). ysis, a mathematical approach that essentially organizes the data Abbreviations: OGFR, opioid growth factor receptor; TU, tumor; INF, infiltrate; Ϫ, no expression; Ϯ, single cell positivity; ϩ,5–35% of by grouping genes with a similar expression pattern, yielded two the cells positive; ϩϩ,35–70% cells positive; ϩϩϩ, Ͼ70% cells major array clusters (i.e., cell lines and skin samples) with this positive; Px, basal cell carcinoma sample not included in the microarray set of 1305 differentially expressed genes and, within each analysis due to unsatisfactory RNA quality; MB, M. Bowen sample. group, a subcluster relating to a particular type of the treatment (Fig. 1, A and B). Topical Imiquimod Treatment Preferentially Modu- lates Genes Involved in Immune Response. The vast major- Protein Isolation and Immunoblotting Analysis ity of up-regulated genes consisted of genes involved in differ- After the RNA extraction with TRIzol, proteins were iso- ent aspects of immune response. Given the previous reports lated from the intermediate fraction according to the manufac- showing that imiquimod is a potent IFN-␣ inducer (17–21), we turer’s recommendation. For immunoblotting analysis, protein first focused on this group of genes. Using hierarchical cluster- extracts were electrophoresed using Novex Tris-Glycine gels ing, we compared the expression profiles of the skin samples (Invitrogen AG) and the buffer system of Laemmli and subse- before and after imiquimod treatment with basal cell carcinoma quently transferred to nitrocellulose in 12.5 mM Tris base, 100 cell lines treated with IFN-␣ or imiquimod. The genes that mM glycine in 20% methanol transfer buffer. The membranes group together according to this method are likely to be coregu- were blocked with 10 mM Tris, 150 mM NaCl, 5% nonfat lated and functionally related (22). Fig. 1C shows the results of powdered milk, and 0.25% Tween 20 and incubated with pri- the clustering analysis, revealing that genes up-regulated in mary antibody for 60 min. Primary antibodies were used at the basal cell carcinoma cell lines and keratinocytes by IFN-␣ following concentrations: opioid growth factor receptor, 0.25 treatment solely represent the same group of genes up-regulated ␮g/ml; and actin, 0.2 ␮g/ml (clone I-19-R; rabbit polyclonal by imiquimod treatment of skin basal cell carcinoma. In vivo IgG; Santa Cruz Biotechnology Inc., Heidelberg, Germany). imiquimod induced the expression of different 2Ј,5Ј-oligoadeny- After incubation with the primary antibody, the membrane was late synthetase isoforms, IFN-induced proteins including MxA washed extensively with 10 mM Tris-HCl, 150 mM NaCl, and and MxB protein, as well as STAT1 and STAT2 transcription 0.25% Tween 20 and incubated for 45 min with the secondary factors. By comparing the genes that were down-regulated in antibody coupled to horseradish peroxidase. Finally, the blots basal cell carcinoma cell lines treated with IFN-␣ (and cell line were developed with chemiluminescence (ECL; Amersham controls) and basal cell carcinoma skin lesions treated with Biosciences, Freiburg, Germany). To reprobe the blots with imiquimod, we could define 79 genes expressed in both evalu- other antibody, the membrane was stripped in 62.5 mM Tris- ated groups. Clustering analysis demonstrated that the majority HCl, 2% SDS, and 100 mM ␤-mercaptoethanol for 30 min at of these genes grouped together, confirming that they belong to 50°C. To remove any remaining SDS, the membrane was the same functional category (Fig. 1E). washed three times with PBS and blocked again. Apart from IFN-␣ and IFN-␥, imiquimod induced a whole spectrum of cytokines (IL-1␤), chemokines (RANTES, ecalectin, Tissue Microarray and Immunohistochemistry MIP-1␣, and CXCL11), and cognate receptors [MIP-1␣/ A tissue microarray containing 52 basal cell carcinoma RANTES receptor (CCR1), CCR7 receptor, IL-3 receptor ␣, tissue samples was constructed as described previously (16). IL-15 receptor ␣], some of which are presented in Fig. 2A. Paraffin-embedded tissue and tissue microarray sections, stained In addition to the IFN-␣ cluster, we could define a IFN-␥ with H&E, were evaluated by an experienced dermatopatholo- cluster, namely, genes that are up-regulated by IFN-␥ as a result gist (R. D.) and subsequently stained with antihuman opioid of Th1 shift and the activation of T cells. Fig. 1D shows parts of growth factor receptor antibody raised against the tandem repeat the “infiltrate” cluster containing some of the IFN-␥-induced region of opioid growth factor receptor, a kind gift from Dr. genes involved in antigen processing (TAP-1 and MECL-1) and

Fig. 1 Gene expression patterns determined by two-way hierarchical clustering analysis of 1305 genes. Relationships between experimental samples are summarized in the dendrogram, in which the pattern and length of the branches reflect the relatedness of the samples. Red indicates a high relative level of expression, whereas green represents a low relative level of expression. A, within the “cell lines” cluster, two major clusters aredistinguishable:

the formation of immunoproteasome (PA28), function of acti- (Fig. 4A). It is of note that the irregular nests of atypical, vated T cells (granzyme B, LAG-3, and T-cell immune regulator neoplastic keratinocytes in M. Bowen exhibited opioid growth 1), and autoregulatory apoptosis in T cells (tumor necrosis factor receptor immunoreactivity before treatment (Fig. 4C). factor receptor 2). To further differentiate whether imiquimod induces opioid This cytokine boom is likely responsible for the influx of growth factor receptor directly or via IFN-␣, we analyzed opioid immune cells observed in histology (Fig. 4, EϪG), which is growth factor receptor expression in basal cell carcinoma cell reflected in the up-regulation of genes representing receptors lines (keratinocytes) treated with imiquimod and IFN-␣ for 24 h (e.g., killer cell inhibiting receptors and killer cell activating by real-time PCR. IFN-␣ up-regulated opioid growth factor receptors, Toll-like receptors, and HLA molecules) pertinent to receptor expression in two basal cell carcinoma cell lines (L1 these cells (summarized in Fig. 2B). Topical imiquimod treat- and L2) and keratinocyte controls (t test, P Ͻ 0.05), whereas ment resulted in an enhanced expression of molecules related to two cell lines demonstrated no significant change in opioid dendritic cell development, such as CD1d, flt-3 ligand, and growth factor receptor expression (Fig. 3C). After treatment Toll-like receptor-3 (Fig. 2, B and C). Moreover, imiquimod with imiquimod, there was no significant change in opioid seems to be a very potent inducer of costimulatory molecules growth factor receptor expression in any of the cell lines inves- such as CD86, CD40, ICAM-2 (CD102), or SLAM (CDw150; tigated. Immunoblotting analysis of basal cell carcinoma cell Fig. 2C). Infiltrating T cells seem to be activated and fully culture extracts demonstrated an increase in opioid growth fac- armed to kill, which is reflected in the up-regulation of cytotoxic tor receptor protein after IFN-␣ treatment (Fig. 3D). We ob- (cytolytic) molecules such as granzyme B and granulysin (Fig. served no changes in opioid growth factor receptor protein 2D). There was also an up-regulation of genes involved in expression after imiquimod treatment of basal cell carcinoma respiratory burst and production of reactive oxygen species in cell lines. phagocytes (neutrophils and macrophages), i.e., neutrophil cy- Opioid Growth Factor Receptor Expression in Basal tosolic factors 1 and 4 (Fig. 2D). Cell Carcinomas Treated with Radiotherapy Is Associated Imiquimod Treatment Up-Regulates Opioid Growth with Longer Recurrence-Free Time. Using a tissue microar- Factor Receptor Expression: An Effect of Interferon-␣? ray containing 27 basal cell carcinomas of aggressive, sclerosing One molecule within the infiltrate cluster with no clear func- type and 25 basal cell carcinomas of nonaggressive type (14 tional relation to this cluster drew our attention. We observed nodular and 11 superficial basal cell carcinomas), we demon- posttreatment induction of opioid growth factor receptor gene strate opioid growth factor receptor expression by tumor cells in expression in skin basal cell carcinomas (2.5-fold increase; P ϭ 23 of 52 (44.2%) evaluated cases (Fig. 4I). Opioid growth factor 0.005). Quantitative real-time PCR analysis confirmed microar- receptor tissue microarray immunohistochemistry results are ray results, demonstrating up-regulation of opioid growth factor presented in Table 2A. Opioid growth factor receptor was pre- receptor expression in six of seven treated skin samples (one dominantly expressed in aggressive, sclerosing basal cell carci- remained unchanged), as shown in Fig. 3A. Normalization of noma (16 of 23 opioid growth factor receptor-positive cases; opioid growth factor receptor to PTCH gene expression (defined Spearman’s ␳ϭϪ0.294; P ϭ 0.035). Nonaggressive basal cell as basal cell carcinoma-specific) again revealed an increase in carcinomas displayed opioid growth factor receptor expression opioid growth factor receptor expression in five of six tested in seven cases (four nodular and three superficial basal cell skin samples (Fig. 3B). Immunohistochemistry using the anti- carcinomas, respectively). In a group of 36 primary basal cell body raised against the immunogenic opioid growth factor re- carcinomas, 13 of which have recurred after radiotherapy, opi- ceptor tandem repeat region revealed an impressive opioid oid growth factor receptor expression was detected in 12 of 23 growth factor receptor up-regulation in all skin basal cell car- basal cell carcinomas that showed no recurrence (Table 2B). cinoma after imiquimod treatment (Table 1 and Fig. 4), includ- The absence of opioid growth factor receptor expression in the ing one M. Bowen sample. In addition to tumor cells, infiltrating primary tumor correlated with the history of recurrence after cells also up-regulated opioid growth factor receptor expression radiotherapy (Spearman’s ␳ϭϪ0.457; P ϭ 0.005). Moreover, (Fig. 4, DϪG). Before treatment, basal cell carcinoma tumor primary basal cell carcinomas that did not recur during the cells demonstrated either weak or no opioid growth factor follow-up period exhibited stronger opioid growth factor recep- receptor immunoreactivity (Fig. 4, B and D). Normal epidermis tor expression than the ones that have recurred, irrespective of demonstrated a nuclear pattern of opioid growth factor receptor the histology type (Mann-Whitney U test, P ϭ 0.020). In pa- expression in the cells from the basal to the granular cell layer tients whose basal cell carcinomas recurred, opioid growth

basal cell carcinoma cell lines and keratinocyte controls. One group of samples initially considered as a basal cell carcinoma cell line (L4), which was eventually overgrown by keratinocytes, clustered together with keratinocyte control. Within the “basal cell carcinoma skin samples” array cluster, two major clusters are distinguishable: the samples before and after (red letters) topical imiquimod treatment. B, moreover, one pretreatment M. Bowen sample (no_MB) clustered quite peripherally to the basal cell carcinoma samples, reflecting the different biology of this entity. After topical imiquimod treatment, this difference disappeared, grouping this sample (IMIQ_MB) with other basal cell carcinomas. Interestingly, two basal cell carcinoma samples from the same patient (P5-1 and P5-2) remained grouped together irrespective of the treatment. C, group of genes up-regulated by IFN-␣ (“IFN-␣ cluster”). Although IMIQ_P4 clustered together with other basal cell carcinoma samples treated with 5% imiquimod cream, this sample failed to up-regulate IFN-␣-inducible genes. D, genes up-regulated by IFN-␥ belonging to a bigger “infiltrate cluster.” The infiltrate cluster is composed of genes characteristic for immune cells, which are up-regulated on topical imiquimod treatment. E, genes down-regulated by IFN-␣. Most of these genes are encoding for ribosomal proteins. F, a cluster of apoptosis genes down-regulated in skin samples after imiquimod treatment. G, the down-regulation of melanocytic markers after topical imiquimod treatment.

Fig. 2 Genes modulated by imiquimod treatment of the skin basal cell carcinoma. Bars represent the posttreatment fold change of the mRNA level of a particular gene when compared with the status before treatment. Black bars indicate up-regulation, whereas white bars represent gene down-regulation. Every gene is represented by gene name and GenBank accession number.

Fig. 3 The expression of opioid growth factor receptor mRNA assessed by real-time PCR (LightCycler). A, opioid growth factor receptor expression in skin basal cell carcinoma before and after topical imiquimod treatment. Results are presented as glyceraldehyde-3-phosphate dehydrogen- ase-normalized ratios to com- pensate for variations in quantity and quality of starting mRNA. Px, basal cell carcinoma sample not included in the microarray analysis due to unsatisfactory RNA quality; MB, M. Bowen sample. B, opioid growth factor receptor mRNA expression in skin basal cell carcinoma normalized to PTCH gene (basal cell carcinoma-specific) to demonstrate tumor-specific opioid growth factor receptor induction. †, due to the high variability of glyceraldehyde-3-phosphate dehydro- genase copy numbers (up to 20- fold difference), these samples were normalized to ␤-actin showing less variability. C, changes in opioid growth factor receptor expression in basal cell carcinoma cell lines and keratinocyte controls after IFN-␣ and imiquimod treatment. D, expression of opioid growth factor receptor protein in basal cell carcinoma cell lines as revealed by immunoblotting analysis. The U937 cell line represents the positive control.

factor receptor expression correlated with a longer recurrence- carcinomas showed down-regulation of additional apoptosis free period (Kaplan-Meier analysis; log-rank test, P ϭ 0.041). genes, among others, the decrease in bcl-2 homolog expression Imiquimod Treatment Affects the Expression of Vari- (Fig. 2F). Some of these genes organized together according to ous Apoptosis Genes. In the skin, imiquimod treatment in- the two-way hierarchical clustering algorithm (Fig. 1G). Down- duced a spectrum of genes implicated in apoptotic signaling and regulation of these genes was not observed in any of the treated the execution of apoptosis (Fig. 2F). Clustering analysis re- cell lines. vealed that the up-regulation of these genes was an infiltrate- The Clearance of Skin Basal Cell Carcinoma Is Asso- specific phenomenon encountered exclusively on imiquimod ciated with the Decrease in Expression of Well-Defined and treatment of the skin basal cell carcinoma. Namely, none of the Potentially Novel Genes Implicated in Basal Cell Carcinoma cell lines investigated (treated with either IFN-␣ or imiquimod) Oncogenesis. In the group of down-regulated genes, 63 genes exhibited a similar pattern of expression. were assigned to be involved in oncogenesis according to the Apart from induction, imiquimod-treated skin basal cell known or predicted function (Fig. 2E). In the skin, imiquimod

Fig. 4 Opioid growth factor receptor immunoreactivity in skin basal cell carcinoma. A, the pattern of opioid growth factor receptor expression in normal epidermis showing clear nuclear opioid growth factor receptor immunoreactivity in the basal, spinous, and part of the granular cell layer (original magnification, ϫ20). B, cy- toplasmic opioid growth factor receptor protein expression in skin basal cell carcinoma (patient P5-2 before treatment). Tumor islets are indicated by arrows. Note the nuclear pattern of opioid growth factor receptor expression in neighbor- ing epidermis, which is not present in tumor islets (original magnification, ϫ10). C, opioid growth factor receptor expression in Bowen’s disease (original magnification, ϫ40). D, absence of opioid growth factor receptor expression in skin basal cell carcinoma (patient 4 before treatment; original magnification, ϫ10). EϪG, massive up-regulation of opioid growth factor receptor immunoreactivity in skin basal cell carcinoma after imiquimod treatment (patients Px, P1, and P2, respectively; original magnification, ϫ10). For better discrimination of the infiltrate, tumor borders are accentuated by a dotted black line. H, nuclear opioid growth factor receptor expression in basal cell carcinoma tumor cells after treatment (original magnification, ϫ100). I, tissue samples from the basal cell carcinoma tissue microarray stained with antibody against the immunogenic opioid growth factor receptor tandem repeat region (original magnification, ϫ10).

treatment resulted in a decrease in expression of Patched regulation of GLI3 in five of six tested samples (Fig. 5). In (PTCH), GLI1, and GLI3 genes, which are implicated in basal addition to these genes, we observed a down-regulation of genes cell carcinoma pathogenesis (23). Real-time PCR analysis reported to be expressed in other human epithelial cancers, such showed down-regulation of PTCH in five of six tested samples, as frizzled (FZD)-7 (24), and c-erb-B2 (HER2). Because the GLI1 down-regulation of four of six tested samples, and down- expression of C-erb-B2 (HER2) has been reported previously in

Table 2 OGFR immunohistochemistry results on a BCC tissue microarray A. OGFR expression with respect to BCC histology in 52 samples in the TMA Histology type OGFR expression* Sclerosing BCC Nodular BCC Superficial BCC ⌺ No 11 10 8 29 Single cell positivity 6 0 2 8 Yes 10 4 1 15 ⌺ 27 6 11 52 B. OGFR expression in primary BCCs that recurred after radiotherapy Histology type Recurrence OGFR expression* Sclerosing BCC Nodular BCC Superficial BCC ⌺ No No 1 4 6 11 Single cell positivity 3 0 2 5 Yes 4 2 1 7 ⌺ 86923 Yes No 5 5 2 12 Single cell positivity 1 0 0 1 Yes 0 0 0 0 ⌺ 65213 NOTE. The 52 samples in the TMA included 36 primary BCCs and 16 secondary BCCs. Abbreviations: OGFR, opioid growth factor receptor; BCC, basal cell carcinoma; TMA, tissue microarray. * Expression refers to tumor cells.

basal cell carcinoma (25), we assessed the expression of FZD7 these genes, one third were up-regulated after imiquimod appli- by real-time PCR. All basal cell carcinoma cases investigated cation, comprising mostly genes that were functionally involved demonstrated FZD7 expression. After imiquimod treatment, in the immune response. We first focused on genes modulated there was a down-regulation of FZD7 gene expression in five of by IFN-␣, knowing that the regression of viral warts or high- six treated skin lesions (Fig. 5). grade intraepithelial neoplasia was associated with increased On the other hand, a group of genes (tyrosinase, Pmel-17/ production of IFN-␣, IFN-␥, and 2Ј,5Ј-oligoadenylate synthe- gp100, Melan-A/MART-1, and p15/MAAT-1) involved in mela- tase (18, 19, 21). In addition to these, imiquimod treatment of nin biosynthesis and known as melanoma-associated antigens the skin basal cell carcinoma induced many other IFN-␣-regu- was singled out (Fig. 2E). The expression of these genes, al- lated genes [various IFN-induced proteins, STAT1, STAT2, though not detectable in any of the basal cell carcinoma cell MxA, and MxB protein (27, 28)]. Topical imiquimod treatment lines or keratinocytes, was down-regulated in skin lesions on resulted in an IFN-␥ increase as well and in the induction of imiquimod treatment (Fig. 1H). Immunohistochemical staining molecules preferentially regulated by this IFN type. showed that the melanocytes were the cells accounting for Apart from IFNs, imiquimod treatment induced the expres- tyrosinase, gp100, and MART-1 expression in basal cell carci- sion of various cytokines, of which IL-1␤ and MIP-1␣ were noma tissue sections. After imiquimod treatment, the melano- recently shown to be up-regulated in macrophages treated with cyte density decreased, which is probably the reason for the S-28463, an imiquimod analog (29). In patients with genital decrease in expression of these markers (data not shown). warts, the erythema at the treatment site correlated with IFN-␥ and RANTES mRNA expression (18), which were up-regulated DISCUSSION in our samples as well. Immune response modifiers are substances that directly or Early infiltrate developing within 3–5 days after treatment indirectly (through the modification of one or more components initiation appears to be of lymphoid/myeloid origin, which is of the immunoregulatory network) affect a specific immune reflected in the up-regulation of genes encoding for different function (26). Imidazoquinolone compounds, of which imi- receptors and cell surface markers expressed in these cells. quimod is the best characterized to date, are such agents. Imi- According to the expression of genes (e.g., IFN-␥, tumor ne- quimod is found to be efficacious in clearing superficial basal crosis factor receptor 2, CD97, ICAM-2, and granzyme B) cell carcinoma (2–4). However, its exact mechanism of action previously described to be expressed in differentiated Th1 cells has yet to be fully understood. To more closely define and (30), imiquimod treatment effectively induced biasing of naı¨ve elucidate the mechanism(s) leading to tumor clearance in vivo, T cells into Th1 cells. Imiquimod activates macrophages via we examined gene expression profiles of basal cell carcinoma of interaction with Toll-like receptor-7 resulting in the secretion of the skin before and after treatment with 5% imiquimod cream by IL-12 (6, 31), favoring the development of IFN-␥-producing T using Affymetrix oligonucleotide arrays. Of a total of 12,000 cells. Exposure to IL-12 results not only in the increased ex- probe sets, we identified 1,305 that were differentially regulated pression of Th1-specific genes but also in the induction of in the skin basal cell carcinoma on imiquimod treatment. Of additional genes such as CCR1 (MIP-1␣/RANTES receptor),

LAG-3, and SLAM (30), whose up-regulation we noted as well on imiquimod treatment. A particularly interesting finding was the up-regulation of CD1d, whose expression was recently shown on dermal dendritic cells and monocyte-derived dendritic cells (32). Apart from monocytes/macrophages, plasmacytoid dendritic cells represent another important target for Toll-like receptor-7-mediated imiquimod activation (6). Activation of the Toll-like receptors on plasmacytoid dendritic cells results in the expression of costimulatory molecules (e.g., CD40 and CD86) necessary for the appropriate activation of antigen-specific T cells (5). Plasmacytoid cells are characterized by a particular set of cell surface molecules, some of which are quite specific for these cells (IL-3 receptor ␣) and have been up-regulated after imiquimod treatment in our samples (CD32, CD38, CD40, and CD43). IL-3 promotes survival but does not drive the plasmacytoid dendritic cells to proliferate (33). Flt3 ligand and granu- locyte colony-stimulating factor, however, have the capability to increase the number of plasmacytoid dendritic cells in vivo (33, 34). Our data show the up-regulation of flt-3 ligand and gran- ulocyte colony-stimulating factor receptor after imiquimod treatment. In view of a recent report demonstrating that imidazoquinolones can directly induce plasmacytoid dendritic cell maturation (35), it is highly suggestive that in addition to activating these cells, imiquimod treatment induces a microenvi- ronment promoting survival and maturation of plasmacytoid dendritic cells. The arrays that we used (HG-U95Av2) contain only Toll-like receptor-1, -2, -3, -5, and 6 genes. Nevertheless, we observed the increase in Toll-like receptor-2 (myelomono- cytic cells, neutrophils, lymphocytes) and -3 [predominantly dendritic cells (36)] transcripts that correlated with the infiltrate characteristics observed by histology. The posttreatment up-regulation of opioid growth factor receptor in the skin (on both the mRNA and protein level) represents a novel and intriguing finding. Opioid growth factor belongs to the group of endogenous opioid peptides and acts as negative regulator of cell proliferation, in addition to its role in pain modulation/transmission (37). Opioid growth factor action is targeted to DNA synthesis, with no effect on the apoptotic pathway (38). Opioid growth factor exerts its action through opioid growth factor receptor, which differs markedly from classical opioid receptors in terms of its function (growth), distribution (neural and nonneural), subcellular location (nu- cleus), and competitive inhibitory profile (37). Zagon et al. (39) demonstrated constitutive expression of opioid growth factor receptor in normal skin, suggesting its regulatory role in DNA synthesis in the basal cell layer. In addition, McLaughlin et al. (40) showed decreased opioid growth factor receptor protein expression in squamous cell carcinoma of the head and neck as compared with normal epithelium, unleashing tumor cells from growth control. Opioid growth factor receptor transcriptional levels, however, remained unaffected, which led the authors to conclude that the changes responsible for decreased opioid growth factor receptor expression are most likely due to trans- Fig. 5 Expression of basal cell carcinoma-associated tumor antigens as- lational/posttranslational defects (40). Using real-time PCR, we sessed by real-time PCR (LightCycler) before and after topical imiquimod could demonstrate opioid growth factor receptor mRNA expres- treatment. Results are presented as glyceraldehyde-3-phosphate dehydro- genase-normalized ratios. Px, basal cell carcinoma sample not included in sion in skin basal cell carcinomas. Our findings confirmed the the microarray analysis due to unsatisfactory RNA quality; MB, M. Bowen opioid growth factor receptor expression pattern described pre- sample. viously by Zagon et al. (39) in normal skin and, furthermore,

revealed weak or absent immunoreactivity in basal cell carci- regulation of genes involved in the hedgehog signaling pathway noma tumor cells. After immunomodulatory treatment with (PTCH, GLI1, and GLI3) implicated in the pathogenesis of imiquimod, we describe up-regulation of opioid growth factor sporadic and hereditary basal cell carcinoma (23). Moreover, we receptor protein in tumor by using the antibody raised against demonstrated expression and down-regulation of another gene the opioid growth factor receptor tandem repeat region. In involved in Wnt signaling, FZD7, which is oncogenic in other addition, we show in vitro that opioid growth factor receptor epithelial cancer types (24, 48). Apart from alterations in the up-regulation in keratinocytes and basal cell carcinoma cell sonic hedgehog pathway and some members of the Wnt cascade lines is IFN-␣-mediated. These findings correspond to in situ (23), there is little available information on other genes involved up-regulation of opioid growth factor receptor shown by immu- in basal cell carcinoma tumor formation. Therefore, high- nohistochemistry. Two recent reports by Wu et al. (41) and throughput microarray technology might be a good tool to Mollick et al. (42) imply that opioid growth factor receptor define and investigate novel basal cell carcinoma tumor anti- protein itself could be immunogenic by demonstrating antigens. opioid growth factor receptor responses in cancer patients after In conclusion, our study using high-density oligonucleotide immunotherapy. Opioid growth factor receptor protein structure arrays provides for the first time a detailed insight into a com- is characterized by the presence of tandem repeats that share plex set of in vivo changes after basal cell carcinoma treatment homology with the extracellular domain of MUC1 (42). It is with imiquimod cream. We have summarized and confirmed the conceivable that through up-regulation of opioid growth factor existing literature evidence on imiquimod mechanism of action receptor, imiquimod might not only be suppressing tumor in one go. Our approach revealed for the first time the up- growth but also increasing tumor immunogenicity. Induction of regulation of opioid growth factor receptor by imiquimod, im- opioid growth factor receptor in infiltrating cells might be a part plying that this effect is likely IFN-␣-mediated. Future studies of the local immune response control as well as an additional will provide more evidence on the role of opioid growth factor signal to produce specific cytokines (Th1/Th2 switch) or anti- receptor as a tumor antigen in basal cell carcinoma and other bodies analogous to other opioid receptors (43). An additional cancer types. positive side affect of opioid growth factor receptor up-regulation might be analgesia despite substantial tissue damage that ACKNOWLEDGMENTS develops locally. In a large series of more than 600 patients with We acknowledge the expert technical assistance of Christa Dudli anogenital warts treated with imiquimod, patients preferred imi- and Beatrice Mu¨ller in immunohistochemistry. We are indebted to quimod 5% cream over other therapies due to the lack of Jessica C. Hassel for her optimized Western blot protocol. We thank associated pain (44). Using a tissue microarray containing a Philomena Selvam for excellent work in cloning of the PCR products. panel of different basal cell carcinoma subtypes, we demonstrate opioid growth factor receptor expression in almost half of the REFERENCES evaluated tumors, primarily those with an aggressive phenotype. 1. Urosevic M, Dummer R. 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Mirjana Urosevic, Patrick A. Oberholzer, Tanja Maier, et al.

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