Peroxisome Proliferator-Activated Receptor ; Is Highly Expressed In

Imaging, Diagnosis, Prognosis

Peroxisome Proliferator-Activated Receptor ; Is Highly Expressed in Pancreatic Cancer and Is Associated With Shorter Overall Survival Times Glen Kristiansen,1Juliane Jacob,1Ann-Christin Buckendahl,1Robert Gru« tzmann,3 Ingo Alldinger,3 Bence Sipos,4 Gu« nter Klo« ppel,4 Marcus Bahra,2 Jan M. Langrehr,2 Peter Neuhaus,2 Manfred Dietel,1and Christian Pilarsky3

Abstract Purpose: Peroxisome proliferator-activated receptor g (PPARg) is a ligand-activated transcription factor that has been implicated in carcinogenesis and progression of various solid tumors, including pancreatic carcinoma.We aimed to clarify the expression patterns of PPARg in pancreatic ductal carcinomas and to correlate these to clinicopathologic variables, including patient survival. Experimental Design: Array-based expression profiling of 19 microdissected carcinomas and 14 normal ductal epithelia was conducted. Additionally,Western blots of pancreatic cancer cell lines and paraffinized tissue of 129 pancreatic carcinomas were immunostained for PPARg.For statistical analysis, Fisher’s exact test, m2 test for trends, correlation analysis, Kaplan-Meier analysis, and Cox’s regression were applied. Results: Expression profiles showed a strong overexpression of PPARg mRNA (change fold, 6.9; P = 0.04). Immunohistochemically, PPARg expression was seen in 71.3% of pancreatic cancer cases. PPARg expression correlated positively to higher pTstages and higher tumor grade. Survival analysis showed a significant prognostic value for PPARg, which was found to be independent in the clinically important subgroup of node-negative tumors. Conclusions: PPARg is commonly up-regulated in pancreatic ductal adenocarcinoma and might be a prognostic marker in this disease. Both findings corroborate the importance of PPARg in tumor progression of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) confers one of the that results in a short gain of patient survival time (2, 3). Most highest mortality rates in malignant human tumors. In 2005, chemotherapeutic regimens target the proliferative potential of the estimated incidence for this disease in the United States tumor cells, which is initially effective in many instances alone reached 32,180 cases, with an equally high rate of deaths because uncontrolled proliferation is a hallmark of these cells. related to this disease (1). The high disease-specific mortality is To specifically target other properties of tumor cells, a variety of due to the fact that pancreatic carcinomas lack early symptoms. candidate genes are being screened for their importance to Consequently PDACs are often detected when the local tumor tumor biology and eligibility as target genes for therapy. One of extent is high and metastatic spread to regional lymph nodes or these candidates is peroxisome proliferator-activated receptor g to distant organ sites has already occurred. Therefore, only (PPARg). PPARg is a member of the nuclear receptor family of f20% of patients are eligible for radical surgery. To date, no ligand-activated transcription factors of which three subtypes satisfactory progress in the treatment of pancreatic adenocarci- (a, h, and g) have been identified thus far, which show nomas has been made and gemcitabine is the only substance different tissue distributions and ligand specificities (4). PPARg was initially recognized as a key regulator of adipogenic differentiation and glucose homeostasis. Recent data supported evidence for participation of PPARg in the biological mecha- Authors’ Affiliations: 1Institute of Pathology and 2Department of Surgery, Charite¤ nisms underlying the carcinogenic evolution, by affecting University Hospital, Berlin, Germany; 3Department of Visceral, Thoracic, and proliferation and differentiation of cancer cells in vitro and Vascular Surgery, University Hospital Dresden, Dresden, Germany; and 4Institute of in vivo (5, 6). PPARg expression has been described in a variety Pathology, University of Schleswig-Holstein Campus Kiel, Kiel, Germany of human malignancies, including, among others, breast Received 4/4/06; revised 7/26/06; accepted 8/24/06. cancer, prostate cancer, glioblastoma cell lines, non–small cell Grant support: Deutsche Krebshilfe (70-2937-SaI). The costs of publication of this article were defrayed in part by the payment of page lung carcinoma, ovarian cancer, and pancreatic carcinoma charges. This article must therefore be hereby marked advertisement in accordance (7–13). To our knowledge, a detailed description of PPARg with 18 U.S.C. Section 1734 solely to indicate this fact. expression in a larger cohort of clinically characterized pan- Requests for reprints: Glen Kristiansen, Institute of Pathology, Charite¤ University creatic cancer cases is still missing. Therefore, we aimed to Hospital, Schumannstrasse 20/21,10117 Berlin, Germany. Phone: 49-30-450-53- 6145; Fax: 49-30-450-53-6945; E-mail: [email protected]. investigate the expression of PPARg in a collection of pancreatic F 2006 American Association for Cancer Research. carcinomas and to correlate the data with clinicopathologic doi:10.1158/1078-0432.CCR-06-0834 variables, including patient survival.

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Table 1. Clinicopathologic variables and PPARg expression of the tumor cohort

Characteristics All cases, n (%) PPAR; low, n (%) PPAR; high, n (%) P All cases 79 (61.2)50 (38.8) Age at diagnosis (y) V64 73 (56.6)44 (34.1) 29 (22.5)0.856 >64 56 (43.4)27 (27.1) 16 (16.3) Tumor stage

pT1 5 (3.9)3 (3.2) 2 (1.6)0.021* pT2 40 (31.0)30 (23.3) 10 (7.8) pT3 78 (60.5)45 (34.9) 33 (25.6) pT4 6 (4.7)1 (0.8) 5 (3.9) Nodal status

pN0 46 (36.5)31 (24.6) 15 (11.9)0.349 pN1 80 (63.5)47 (37.3) 33 (26.2) Grade

G1 10 (7.8)7 (5.4) 3 (2.3)0.118* G2 66 (51.2)44 (34.1) 22 (17.1) G3 53 (41.1)28 (21.7) 25 (19.4) Residual tumor status

R0 94 (82.5)58 (50.9) 36 (31.6)0.452 R1 20 (17.5)10 (8.8) 10 (8.8)

*m2 for trends.

n Materials and Methods nantly localized in the head of the pancreas ( = 118; 91.5%), four tumors were in corpus/cauda region, and in seven cases, the papilla duodeni major was involved. The stage of tumors was assessed Patients. The study was carried out with institutional review board according to International Union Against Cancer. The clinicopathologic consent using surgically resected tissue of 129 patients with PDAC. Of characteristics of the tumor cohort are described in Table 1. these, 95 cases had surgery in Berlin between the years 1991 and 2000, Cell lines. Pancreatic cancer cell lines were obtained from American whereas 34 cases underwent surgery in Dresden. Additionally, frozen Type Culture Collection: (Wesel, Germany); AsPC-1 was originally tissue from 33 patients undergoing pancreatic surgery was included for isolated from ascites of a patient with a G2 PDAC. Mia Paca-2, PANC1, expression profiling. The study group for immunohistochemistry and PancTuI stemmed from weakly differentiated (G3) primary PDACs. consisted of 70 male and 59 female patients whose age ranged from Capan-1 was isolated from a lymph node metastasis of a PDAC 34 to 80 years at the time of diagnosis (median, 64). Complete follow- patients, and Colo357 and Panc89 were from liver metastases. up data were available for all cases. The overall survival time ranged Expression profiling. We analyzed the expression of microdissected from 2 to 492 weeks (median, 72). One hundred two patients died PDACs from 19 patients and microdissected normal duct cells from during follow-up after a median overall survival time of 55 weeks. 14 patients undergoing pancreatic surgery for nonmalignant diseases, Tumor histology was determined according to the criteria of the as described before. Briefly, freshly frozen tissue samples of PDAC were WHO. All cases were ductal adenocarcinomas, which were predomi- obtained from surgical specimens of patients who were operated at the Department of Visceral, Thoracic, and Vascular Surgery, Carl Gustav Carus, Technical University of Dresden (Dresden, Germany), and the Department of General Surgery, University of Kiel (Kiel, Germany) between 1996 and 2003. Normal pancreatic tissue was obtained from 14 patients who underwent pancreatic resection for other pancreatic diseases. These tissues were histologically normal tissues without dysplastic changes in the ducts and were taken from the distal parts of the resected pancreas. PDAC cells and normal ductal cells were dissected manually using a sterile injection needle. The estimated cellularity was 10,000 to 11,000 cells per microdissected sample. The cellularity of the dissections was f95%. The RNA was extracted, amplified, and hybridized to the U133 GeneChip set as described previously (14). The obtained data were normalized and condensed using the dChip software,5 which also was used to generate the heatmap. Identification of differential expression was done using the SAM software.6 Genes were assigned as differential expressed if the fold change was larger than two and the q value was below 5% resulting in a set of differentially expressed genes for PDAC. Pathway analysis of genes interacting with PPARg was done using PathwayAssist 3.0 (Stratagene, Amsterdam, The Netherlands). Such genes were used

Fig. 1. Box plot of arbitrary fluorescent units of PPARg expression in microdissected pancreatic tissue. T, expression in microdissected PDAC; N, expression in microdissected normal duct epithelia. To confirm the differential 5 http://www.dchip.org. expression, t test was applied (P =0.04). 6 http://www-stat.stanford.edu/ftibs/SAM/.

www.aacrjournals.org 6445 Clin Cancer Res 2006;12(21)November 1, 2006 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2006 American Association for Cancer Research. Imaging, Diagnosis, Prognosis to identify PPARg-related genes within the set of differentially primary antibody. Detection took place by the conventional labeled expressed genes. streptavidin-biotin (DAKO) method with alkaline phosphatase as the Western blot of PPARg. Pancreatic cell lines were cultivated in RPMI reporting enzyme according to the manufacturer’s instructions. Fast Red 1640 with 10% FCS (Invitrogen, Karlsruhe, Germany), lysed using (Sigma-Aldrich, Munich, Germany) served as chromogen. Afterwards, Novex LDS sample buffer, and subjected to gel electrophoresis using the the slides were briefly counterstained with hematoxylin and aqueously Novex Nupage system (Invitrogen, Karlsruhe, Germany). After comple- mounted. tion, the proteins were transferred to a nitrocellulose membrane Evaluation of the immunohistochemical stainings. The slides were (Amersham Pharmacia Biosciences, Freiburg, Germany). PPARg was evaluated by two clinical pathologists. Classification of nuclear detected by using the PPARg-specific antibody (E8, Santa Cruz immunoreactivity was based on staining intensity and the amount of Biotechnology, Santa Cruz, CA) diluted 1:100 and the enhanced positive tumor and graded as 0, no staining; 1, weak; 2, moderate; and chemiluminescence plus Western blot detection system (Amersham 3, strong. The additional cytoplasmic staining seen in a few cases was Pharmacia Biosciences). After detection, the blots were stripped and not evaluated. h-actin was detected by using a mouse monoclonal antibody (Abcam, Statistical analysis. The data were compiled with the software Heidelberg, Germany) diluted 1:5,000. The secondary antibody was package SPSS, version 12.0 (Chicago, IL). Fisher’s exact and m2 tests diluted 1:25,000 in both cases. were used to assess the statistical significance of the association between Quantitative reverse transcription-PCR of PPARg in cell lines. For the expression of PPARg and clinicopathologic variables. Correlation determination of PPARg expression in PDAC cell lines, total RNA was analysis comprised Pearson’s analysis for the array data and Spearman’s prepared and total RNA (5 Ag) reverse transcribed was done using analysis for the immunohistochemistry data. Univariate survival anal- SuperScript II reverse transcriptase and random hexamer primers ysis was done according to Kaplan-Meier, and differences in sur- (Invitrogen, Heidelberg, Germany) according to the manufacturer’s vival curves were assessed with the log-rank test. Ps < 0.05 were suggestion. The genes were amplified with the SYBR Green Universal considered significant. All statistics were accredited by the head PCR Master Mix according to the manufacturer’s instructions, with biostatistician of the Tumor Centre, Charite´University Hospital (Berlin, the ABI Prism 5700 Sequence Detection System (Weitenstadt, Germany). Germany) using the following primers: PPARg,5¶-atgacagcgacttggcaata- 3¶ (forward) and 5¶-gaatgtcttcaatgggcttca-3¶ (reverse); h-actin, 5¶-aatgctat- cacctcccctgtgt-3¶ (forward) and 5¶-aagccaccccacttctctctaa-3¶ (reverse). Results Immunohistochemistry. Formalin-fixed paraffin-embedded tissue was freshly cut into slices of 4 Am. The sections were mounted on Expression profiling Superfrost slides (Menzel Gla¨ser, Braunschweig, Germany), dewaxed To analyze the expression of PPARg in PDAC, we evaluated with xylene, and gradually hydrated. Antigen retrieval was achieved by the obtained gene expression profiles from microdissected pressure cooking in 0.01 mol/L citrate buffer for 5 minutes. The primary samples of normal ductal epithelia and adenocarcinomas. PPARg antibody (E8) was diluted 1:75 using a background reducing dilution buffer (DAKO, Hamburg, Germany). No other blocking agents PPARg was overexpressed in ductal adenocarcinomas by a P were used. The primary antibody was incubated at room temperature factor of (median) 6.92 ( = 0.04; Fig. 1). We were then for 1 hour. As a negative control, two slides were processed without interested if target genes of PPARg or genes regulating PPARg

Table 2. PPARg-associated genes differentially expressed in PDAC identified using gene expression analysis of microdissected samples

Probe set ID Symbol DescriptionF/C (T/N) 208510_s_at PPARG Peroxisome proliferative-activated receptor, g 6.09 Regulating PPARg activity 204832_s_at BMPR1A Bone morphogenetic protein receptor, type IA 5.45 209478_at STRA13 Stimulated by retinoic acid 13 homologue 3.00 201694_s_at EGR1 Early growth response 1 0.53 226705_at FGFR1 Fibroblast growth factor receptor 1 0.49 203973_s_at CEBPD CCAAT/enhancer binding protein, y 0.47 206254_at EGF Epidermal growth factor (h-urogastrone)0.21 205290_s_at BMP2 Bone morphogenetic protein 2 0.14 Regulated by PPARg 201884_at CEACAM5 Carcinoembryonic antigen-related cell adhesion molecule 5 4.03 208795_s_at MCM7 MCM7 minichromosome maintenance deficient 7 2.58 209173_at AGR2 Anterior gradient 2 homologue 2.26 203752_s_at JUND Jun D proto-oncogene 0.49 228806_at RORC Retinoic acid receptor-related orphan receptor C 0.47 207720_at LOR Loricrin 0.47 205357_s_at AGTR1 Angiotensin II receptor, type 1 0.41 208343_s_at NR5A2 Nuclear receptor subfamily 5, group A, member 2 0.36 208711_s_at CCND1 Cyclin D1 2.82 Binding partner of PPARg 206074_s_at HMGA1 High mobility group AT-hook 1 2.49 206410_at NR0B2 Nuclear receptor subfamily 0, group B, member 2 0.47 202426_s_at RXRA Retinoid X receptor, a 1.04

NOTE: Genes were classified using the Pathway Assist software [F/C (T/N)fold change of tumor above normal tissue]. For completeness, the fold change of the PPARg coactivator retinoid X receptor a is also shown.

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Fig. 2. Expression of PPARg and related genes in PDAC. A, PPARg pathway containing PPARg target genes in the lower part and genes that regulate PPARg function in the upper part. Left of PPARg,the coactivators of PPARg. CCND1is depicted on the right of PPARg because it appears that it regulates and is regulated by PPARg (green, underexpressed; red, overexpressed; blue, not differentially expressed; +, positive regulation; À, negative regulation). B, heatmap of PPARg-related genes (red,highexpression; blue, low expression).

function were also differentially expressed. These genes were receptor a was not differentially expressed in PDAC (Fig. 2A identified by the PathwayAssist program. Of the eight identified and B; Table 2). target genes, (CEACAM5, MCM7, AGR2, JUND, AGTR1, LOR, NR5A2, and RORC) three are also overexpressed in pancreatic Western blot and quantitative reverse cancer (CEACAM5, MCM7, and AGR2), whereas the others transcription-pCR are underexpressed. Of seven genes known to regulate PPARg Western blot of PPARg with the E8 antibody revealed a function (STRA13, EGR1, CEBPD, BMPR1A, BMP2, EGF, and specific band between 50 and 60 kDa, as expected (Fig. 3A). FGFR1), the activator BMPR1A and the inhibitor STRA13 Expression of PPARg protein corresponded well with the were up-regulated in PDAC, whereas the others were down- expression levels of PPARg mRNA as seen on the microarrays regulated (Table 2). In a stratified correlation analysis of (Fig. 2) and CT values of the reverse transcription-PCR PPARg with these 15 genes in normal and tumor tissue, we (Fig. 3B). This indicates that the differences in expression seen found quite divergent results: in normal tissue, PPARg was in the primary tissue is represented in pancreatic cancer cell significantly correlated with CEACAM5 [correlation coefficient lines and also shows that the RNA and protein level of PPARg (cc), 0.997; P < 0.001], MCM7 (cc, 0.740; P =0.002),JUND are correlated. (cc, À0.725; P = 0.002), STRA13(cc, 0.784; P = 0.001), EGR1 (cc, À0.570; P = 0.026), CEBPD (cc, À0.556; P = 0.032), PPAR; immunostaining in pancreatic tissue BMPR1A (cc, 1; P < 0.001), and NR0B2 (cc, À0.526; P = 0.044). To validate the observed overexpression of PPARg in PDAC, In tumor tissue, PPARg correlated only to CEACAM (cc, 0.478; we did immunohistochemistry using a mouse monoclonal P = 0.033), JUND (cc, 0.554; P = 0.011), and FGFR1 (cc, 0.449; antibody. Within normal pancreatic acinar epithelium, staining P = 0.047). This discrepancy of different correlations in for PPARg was not detected. Normal ductal epithelium was normal and malignant tissue suggests an impaired function of mostly PPARg negative or showed a very weak nuclear or PPARg in tumor tissue. The coactivator of PPARg retinoid X cytoplasmic staining (Fig. 4A). Intraductal proliferations with

www.aacrjournals.org 6447 Clin Cancer Res 2006;12(21)November 1, 2006 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2006 American Association for Cancer Research. Imaging, Diagnosis, Prognosis atypia diagnostic of pancreatic intraepithelial neoplasia (PanIN) occasionally exhibited a nuclear pronounced PPARg expression (Fig. 4B). In invasive carcinomas, a heterogenous nuclear immunoreactivity was noted, which reached from weak to strong (Fig. 4C and D). In total, 71.3% of carcinomas were PPARg positive, 28.7% of cases were PPARg negative. The positive carcinomas were further classified into weak (32.6%), moderate (25.6%), and strong (13.2 %) expression of PPARg. For statistical analysis, we grouped carcinomas, which had either no or a weak PPARg expression together (low level group, 61.3% of cases) and opposed them to cases with a moderate or strong expression of PPARg (high level group, 38.7% of cases). In cross-tables, we did not find any significant association of PPARg staining intensity with patient age, nodal status, lymphangiosis or hemangiosis carcinomatosa, and histologic grade. However, there is a significant association of higher PPARg expression levels with higher pT stages (Table 1). However, Spearman’s rank correlations showed a significant correlation of PPARg immunoreactivity with tumor grade and pT stage (Table 3). Fig. 4. PPARg immunohistochemistry. A, normal pancreatic parenchyma. No significant PPARg expression is seen in normal ductal epithelium (central), PPAR; expression and patient survival whereas acinar epithelia exhibit a very weak cytoplasmic staining. B, moderate Univariate survival analysis. In univariate survival analyses, nuclear PPARg expression in atypical epithelia of a PanIN lesion. C and D, moderate/strong nuclear PPARg expression in pancreatic adenocarcinoma. cumulative survival curves were calculated according to the Kaplan-Meier method. Differences in survival were assessed with the log-rank test. The conventional prognostic markers analysis of PPARg expression stratified for pT stage, tumor histologic tumor grade, nodal status, and patient age, but also grading, and nodal status. In the subgroup of pT3/pT4 tumors, PPARg immunoreactivity reached significance for different high levels of PPARg expression were associated with shorter overall survival times (Fig. 5A; Table 4). No significant overall survival times (median survival time, 97 versus 56 differences in survival time became apparent for patient gender weeks; P = 0.001; Fig. 5B), which was not found in the group of or pT stage. pT1/pT2 tumors (P = 0.243). In the subgroup of G1/G2 tumors, high levels of PPARg expression were associated with shorter Survival analysis in patient subgroups overall survival times (median survival time, 128 versus 77 To assess if PPARg might bear a pronounced prognostic effect weeks; P = 0.011; Fig. 5C), which was not found in the group of in patient subgroups, we conducted an extensive Kaplan-Meier G3 tumors (P = 0.726). Stratifying for nodal status, in node- negative cases, survival time was significantly shorter in the group of tumors with high levels of PPARg expression (median survival time, 148 versus 52 weeks; P = 0.012; Fig. 5D), which again was not seen in node positive cases (P = 0.964). Multivariate survival analysis

In the Cox regression model, we included pT stage (pT1/pT2 versus pT3/pT4), tumor grade (G1/G2 versus G3), patient age, nodal status (pN0 versus pN1), and PPARg expression (0/1+ versus 2/3+). In the analysis of all cases, overall survival time was significantly dependent on nodal status, patient age, and tumor grade (Table 5). Analogous to the stratified univariate

Table 3. Correlation of PPARg protein expression in pancreatic cancer with conventional clinicopathologic variables

Statistic pT pN Grading Residual Age Fig. 3. Detection of PPARg by Western blot and quantitative reverse transcription- status status tumor (R) PCR in pancreatic cancer cell lines. A, top lane, PPARg; bottom lane, h-actin, which À served as a loading control. Expression of PPARg protein corresponded well with PPARg cc 0.249 0.132 0.225 0.089 0.119 the expression levels of PPARg mRNA as seen on the microarrays (Fig. 2). P 0.004 0.140 0.010 0.347 0.179 B, expression of PPARg RNA in PDAC cell lines normalized to Capan-1expression in n 129 126 129 114 129 percent. For PPARg, quantitative reverse transcription-PCR was done and the obtained CT values were first normalized to h-actin as housekeeping gene. The obtained DCT values were then normalized to the DCT value from the Capan-1 Abbreviation: n, number of cases. cell line, which was arbitrarily set to 100%.

ClinCancerRes2006;12(21)November1,2006 6448 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2006 American Association for Cancer Research. PPARg in Pancreatic Carcinoma analysis, the Cox regression analysis in the subgroup of node- negative patients (n = 46) revealed that PPARg expression was a significant indicator for shortened patient survival (relative risk, 2.46; P = 0.039) as shown in Table 6.

Discussion

PPARg belongs to the nuclear hormone receptor superfamily that includes the receptors for steroid, thyroid hormone, vitamin D, and retinoid acids (15). Activated by binding of specific ligands, PPARg forms a complex with retinoid X receptors (16). This complex binds to so-called peroxisome proliferator response elements in target genes, which activates transcription. PPARg is expressed at high levels in fat tissue but has also been described in other tissues (e.g., muscle, adrenal gland, and liver; refs. 17–20). PPARg functions in many processes related to cellular development proliferation and differentiation. PPARg is expressed in the majority of malignant human tumors, ranging from hematologic malignancies (21, 22) to solid tumors [e.g., gastric and colon cancer (23, 24), prostate, breast and ovarian cancer (8, 11, 25), non– small cell lung cancer (10), urothelial carcinoma (26), renal cell carcinoma (27), thyroid cancer (28, 29), esophageal squamous cell carcinoma (30), osteosarcoma (31), liposarcoma (32), glioma (33), and neuroblastoma (34)]. Administration of synthetic PPARg ligands (thiazolidindiones) has shown marked antineoplastic effects on various cell lines and tumors. PPARg agonists are antiproliferative, at least in part by up-regulating cyclin-dependent kinase inhibitors and they induce apoptosis in some tumor cell lines (35–37) or differentiation of cells in others (25, 38). This might be explained by the stimulation of overexpressed but inactive PPARg in tumors and might also be the case in PDAC because we observed overexpression of only three typical PPARg target genes in PDAC and also an underexpression of five PPARg target genes. This is also mirrored in the loss of correlations of PPARg with PPARg- associated genes we found in tumor tissue. These data indicate that other signal transduction pathways might be involved in regulating PPARg target gene expression. Interestingly, STRA13, a gene reported to negatively regulate PPARg, is overexpressed in PDAC. This overexpression might result in an inactivation of PPARg and might not be compensated by the overexpression of the PPARg activator BMPR1A resulting in PPARg inactivation. The stock of literature on PPARg in pancreatic cancer thus far focused on cell lines and small tumor cohorts. Eibl et al. (35) found PPARg expressed in six pancreatic cancer cell lines (AsPC-1, BxPC-3, Capan-2, HPAF-II, MIA Paca-2, and PANC1) and could show a decreased cell viability by treatment with PPARg agonists, which was time and dose dependent. Farrow et al. describe PPARg expression in the cell lines AsPC-1, SUIT-2, BxPC-3, and MIA Paca-2 and conducted invasion assays under PPARg agonist treatment. They found a reduced invasiveness and reduced levels of proinvasive factors as tissue plasminogen activator, matrix metalloproteinase 3, matrix

Fig. 5. Survival analysis. Kaplan-Meier analysis of overall survival times stratified according to PPARg expression in patients with pancreatic adenocarcinomas (dotted line, low level; continuous line,highlevels).A, all cases (n =129). B, subgroup of pT3/pT4 cases (n =85).C, subgroup of G1/2 cases (n =53). D, subgroup of node-negative cases (n =46).

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Table 4. Kaplan-Meier survival analysis of PPARg expression and selected tumor variables

Cases Events Median survival (wk) SE P, log-rank test Age at diagnosis (y) V64 73 53 84 8 0.047 >64 43 40 64 7

Tumor stage pT1/pT2 45 35 71 12 0.937 pT3/pT4 84 67 76 8 Nodal status pN0 46 29 96 23 0.001 pN1 80 71 60 5 Grade G1/G2 76 54 84 7 <0.001 G3 53 48 52 6 PPARg expression low 79 58 78 10 0.048 high 50 44 64 12 metalloproteinase 7, and urokinase-type plasminogen activator status was apparent. On survival analysis, higher rates of PPARg (39, 40). Sasaki et al. (12) found PPARg mRNA in five of seven expression where associated with shorter overall survival times pancreatic carcinoma cell lines and also in five of seven cancer were seen, which even reached an independent prognostic samples, whereas all four adjacent normal tissues showed no value in the subgroup of node-negative cases. In the subgroups expression. Functionally, they could show an inhibition of of patients with pT3 stage disease or less aggressive tumors anchorage-independent growth of cell lines. Motomura et al. (G1/G2), PPARg could as well define significant risk groups. described PPARg expression on RNA and protein level in 4 of PPARg might therefore be considered a new candidate marker 4 cell lines and in 9 of 10 pancreatic cancer samples. No of patient prognosis in patients with pancreatic cancer, which expression was found in normal ductal epithelium. Their clearly warrants further study. It seems paradoxical that we immunohistologic analysis clearly showed the nuclear pattern found PPARg overexpressed in more aggressive tumors, that is to be expected of a transcription factor and which we although PPARg ligands are known to decrease tumor growth. found as well (41). In addition, a dose-dependent inhibition of As stated earlier, this could hint at a disrupted PPARg cell growth by troglitazone accompanied by an up-regulation of downstream pathway, which would be endorsed by our array p27 was seen. Because this could be blocked by p27 antisense data that does not show a stringent correlation of PPARg RNA oligonucleotides, the authors conclude that the PPARg effect on with typical PPARg target genes. It also parallels the findings of cell growth is p27 mediated. Itami et al. found PPARg expressed Zhang et al. (11) who reported higher PPARg levels in more in 5 of 5 cell lines and conducted immunohistochemistry on aggressive ovarian cancers. The data on PPARg expression in 47 primary pancreas cancer samples and 15 liver metastases. primary tumors and patient prognosis is sparse and divergent. Seventy-five percent of primary tumors and 80% of metastases Jiang et al. (43) found lower mRNA levels in breast cancer showed a high rate of PPARg expression. Unfortunately, no patients with high-risk tumors. Papadaki et al. (44) investigated further correlation of PPARg with clinicopathologic variables of breast cancer tissues immunohistochemically and found low the primary tumors was investigated (42). rates of PPARg to be prognostic of shorter disease-free survival To our knowledge, our study is the first description of PPARg times. It is of interest to note, however, that although they used expression in a larger cohort of clinically characterized primary the same antibody we used in our study, they reported a pancreatic tumors by immunohistochemistry. We used a predominantly cytoplasmic and even membranous immunos- monoclonal PPARg antibody, which is applicable to paraffi- taining, which they subjected to statistical evaluation. nized tissue and enabled us to analyze clinically characterized Given the wealth of experimental data on the growth- archive material. This antibody is widely used in PPARg inhibitory effect of agonistic PPARg ligands on pancreatic research (23, 28, 36, 42) and yields clear immunohistologic cancer cell lines and the high rate of PPARg expression found in stainings we found reproducible and easy to analyze. We saw pancreatic cancer, we think that this strongly suggests to further PPARg expression in 71.3% of pancreatic carcinoma tissues. investigate the potential of PPARg ligands for treatment of High rates of PPARg expression were noted in 38.7% of pancreatic cancer. This idea is also supported in consideration carcinomas with a clear predominance in larger (pT3) and less of the convenient toxicity profile of these drugs and the very well-differentiated (G3) tumors, still no correlation to nodal limited treatment options that are available for patients with this disastrous disease. Still, there are two caveats. First,

Table 5. Cox regression model, including conventional variables and PPARg expression in Table 6. Cox regression model, including all cases (n = 129) conventional variables and PPARg expression in the subgroup of node-negative cases (n = 46) Overall survival (102 events) Overall survival (29 events) Relative risk 95% CI P Relative risk 95% CI P pT stage 1.061 0.694-1.624 0.784 Grading 1.983 1.307-3.008 0.001 pT stage 0.806 0.374-1.735 0.581 Nodal status 1.864 1.196-2.905 0.006 Grading 1.763 0.787-3.947 0.168 Age 1.026 1.002-1.050 0.033 Age 1.030 0.986-1.076 0.180 PPARg 1.271 0.831-1.944 0.269 PPARg 2.463 1.048-5.788 0.039

ClinCancerRes2006;12(21)November1,2006 6450 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2006 American Association for Cancer Research. PPARg in Pancreatic Carcinoma previous studies with PPARg ligands in liposarcoma as well as possible role of PPARg expression in pancreatic cancer and to in prostate and breast cancer have shown diverse effects elucidate its diagnostic or prognostic role and to clarify the and were only partially successful (32, 45, 46). Also has therapeutic potential of PPARg ligands. troglitazone, the PPARg ligand used in the breast cancer study, meanwhile been withdrawn by the FDA because of hepatic toxicity. Second, there is evidence that PPARg ligands Acknowledgments might even further induce tumor malignancy in some tissues (47–49). In conclusion, further studies are needed to clarify the We thank Britta Beyer for the excellent technical assistance.

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www.aacrjournals.org 6451 Clin Cancer Res 2006;12(21)November 1, 2006 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2006 American Association for Cancer Research. Peroxisome Proliferator-Activated Receptor γ Is Highly Expressed in Pancreatic Cancer and Is Associated With Shorter Overall Survival Times

Glen Kristiansen, Juliane Jacob, Ann-Christin Buckendahl, et al.

Clin Cancer Res 2006;12:6444-6451.

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