PERIPROSTATIC ADIPOSE TISSUE AS A MODULATOR OF PROSTATE CANCER AGGRESSIVENESS

Periprostatic Adipose Tissue as a Modulator of Prostate Cancer Aggressiveness David S. Finley,* Valerie S. Calvert, Junichi Inokuchi, Alice Lau, Navneet Narula, Emanuel F. Petricoin, Frank Zaldivar, Rosanne Santos, Darren R. Tyson† and David K. Ornstein†,‡ From the Departments of Urology (DSF, JI, AL, RS, DKO), Pathology (NN) and Pediatrics (FZ), University of California-Irvine, Orange, California, Center for Applied Proteomics and Molecular Medicine, George Mason University (VSC, EFP), Manassas, Virginia, and Department of Cancer Biology, Vanderbilt University (DRT), Nashville, Tennessee

Purpose: Adipose tissue has been suggested to contribute to the pathogenesis of various disease states, including prostate cancer. We investigated the association of cytokines and growth factors secreted by periprostatic adipose tissue with pathological features of aggressive prostate cancer. Materials and Methods: Periprostatic adipose tissue was harvested from patients undergoing radical prostatectomy and cultured for 24 hours to generate conditioned medium or snap frozen immediately for functional signaling profiling. Multiplex analysis of the periprostatic adipose tissue conditioned medium was used to detect cytokine levels and compared to patient matched serum from 7 patients. Interleukin-6 in serum and periprostatic adipose tissue conditioned medium was further analyzed by enzyme-linked immunosorbent assay and correlated with clinical variables, such as age, body mass index and Gleason score, in 45 patients. Interleukin-6 expression in periprostatic adipose tissue was determined by immunohistochemistry. Reverse phase protein microarray technology was used to analyze cell signaling networks in periprostatic adipose tissue. Results: Interleukin-6 in periprostatic adipose tissue conditioned medium was approximately 375 times greater than that in patient matched serum and levels correlated with pathological grade. This finding was further extended by cell signaling analysis of periprostatic adipose tissue, which showed greater phosphorylation on Stat3 with high grade tumors (any component of Gleason score 4 or 5). Conclusions: Higher Gleason score correlated with high levels of conditioned medium derived interleukin-6. Moreover, cell signaling analysis of periprostatic adipose tissue identified activated signaling molecules, including STAT3, that correlated with Gleason score. Since STAT3 is interleukin-6 regulated, these findings suggest that periprostatic adipose tissue may have a role in modulating prostate cancer aggressiveness by serving as a source of interleukin-6. Also, we found low numbers of inflammatory cells in the fat, suggesting that adipocytes are the major secretors of interleukin-6. Key Words: prostate, prostatic neoplasms, adipocytes, cytokines, intercellular signaling peptides and proteins

Abbreviations and Acronyms AMPK ⫽ adenosine monophosphate-activated protein kinase BMI ⫽ body mass index CM ⫽ conditioned medium CXCL ⫽ CX-chemokine ligand ELISA ⫽ enzyme-linked immunosorbent assay G-CSF ⫽ granulocyte colony stimulating factor IFN ⫽ interferon IL ⫽ interleukin JAK ⫽ Janus kinase MCP-1 ⫽ monocyte chemoattractant protein-1 mTOR ⫽ mammalian target of rapamycin PBS ⫽ phosphate buffered saline PPAT ⫽ periprostatic adipose tissue PSA ⫽ prostate specific antigen STAT ⫽ signal transducer and activator of transcription protein TNF␣ ⫽ tumor necrosis factor-␣ VEGF ⫽ vascular endothelial growth factor

Submitted for publication January 21, 2009. Study received University of California-Irvine institutional review board approval. * Financial interest and/or other relationship with Intercool Therapetutics (Cardium). † Equal study contribution. ‡ Correspondence: Vanguard Urologic Research Foundation, Vanguard Urologic Institute, Texas Medical Center, 4100 Fannin St., Suite 2300, Houston, Texas (e-mail: [email protected]). Supplementary material provided at www.David.Ornsteinmd.com.

See Editorial on page 1255.

0022-5347/09/1824-1621/0 THE JOURNAL OF UROLOGY® Copyright © 2009 by AMERICAN UROLOGICAL ASSOCIATION

Vol. 182, 1621-1627, October 2009 Printed in U.S.A. DOI:10.1016/j.juro.2009.06.015

www.jurology.com

1621

1622

PERIPROSTATIC ADIPOSE TISSUE AND PROSTATE CANCER

OBESITY is implicated as a major risk factor for various human cancers1 and there is now compelling evidence linking obesity with prostate cancer aggressiveness.2,3 Several studies show more adverse pathological features and higher risk for biochemical recurrence in obese men undergoing radical prostatectomy.4 Also, in a large prospective, randomized trial of patients with advanced prostate cancer treated with radiotherapy combined with androgen deprivation increased BMI was an independent risk factor for prostate cancer specific mortality.5 Although it has been theorized that the association of obesity with increasing prostate cancer aggressiveness is the result of endocrine derangements, such as increased serum estrogen, insulin, insulin-like growth factor-1 and leptin, and decreased free testosterone, the underlying biological mechanism to explain this association remains to be determined.6 – 8 Adipose tissue was long thought of merely as a repository of triglycerides available for use when glucose was low but emerging data suggest that adipose tissue is a bioactive endocrine organ that affects energy balance and has an important functional role in modulating human disease. Recently Calvert et al established a link between omental fat and the pathogenesis of nonalcoholic fatty liver disease.9 Furthermore, it was noted that visceral adipose tissue is different from peripheral adipose and may elaborate a unique set of cytokines/growth factors. In addition to the classic adipokines leptin and adiponectin, numerous inflammatory mediators are secreted from adipose tissue, including many ILs (eg IL-1␤, IL-6 and IL-10), chemokines (IL-8 or CXCL8, CCL2 or MCP-1 and CXCL10 or IFN-inducible protein-10) and growth factors (nerve growth factor, VEGF and TNF␣).1 Adipose tissue is an important mediator of local and systemic inflammatory responses.10 Notably about 30% of 1,300 gene transcripts expressed in white adipose tissue encode genes related to inflammation.11 Various cytokines are implicated as mediators of prostate cancer aggressiveness and there have been reports that serum levels of cytokines correlate with prostate tumor aggressiveness. However, the association of increased serum cytokines and prostate cancer aggressiveness has not been consistently found and the source of these cytokines is not known.12 IL-6 is a proinflammatory cytokine that is strongly implicated as a carcinogenesis promoter.13 Increased IL-6 expression is observed in multiple epithelial cancers, including breast, lung, colon, ovarian and prostate cancer.13,14 IL-6 modulates growth and differentiation of malignant tumors, and increased tissue and/or serum levels are associated with poor prognosis in cases of several solid and hematopoietic

neoplasms. 14 Several investigators have suggested the involvement of IL-6 in prostate cancer development and progression.15 Deregulated IL-6 expression was reported in prostate cancer and the levels of this cytokine and its receptor increase during prostate carcinogenesis and progression.16,17 The concomitant increase in receptor and ligand creates a functional autocrine loop.18 Moreover, IL-6 is strongly expressed by prostate cancer cells, such as DU145 and PC-3 cells, and directly contributes to the proliferation of these cells in vitro.19 Serum and plasma IL-6 levels are increased in patients with advanced disease20 and correlate with the tumor burden, as assessed by serum PSA or clinically evident metastasis.21 The association of increased IL-6 serum levels with patient morbidity has made IL-6 a candidate for targeted therapy for metastatic prostate cancer.22 Currently several phase I/II clinical trials for various human cancers are based on targeting IL-6 signaling.10 Further evidence supporting a role for IL-6 in prostate carcinogenesis is the finding that IL-6 receptor expression and STAT3 activation are frequently increased in prostate cancer cases.19,22,23 IL-6 mediates effects on tumor cells primarily by the activation of STAT3, which promotes cell cycle progression, tumor invasion and host immune system evasion.24 Systemic levels of IL-6 and other cytokines/ growth factors may not accurately reflect the local contribution of these factors to prostate cancer development and progression. We examined the hypothesis that PPAT is the source of progression promoting cytokines/growth factors in patients with prostate cancer. We determined whether PPAT elaborates and contributes significantly to local levels of various cytokines, including IL-6. We noted whether the association of increased elaboration of IL-6 and expression of activated (phosphorylated) STAT3 correlate with pathological tumor grade.

METHODS Tissue Procurement and CM Generation This study was initiated after receiving University of California-Irvine institutional review board approval. Clinical and pathological characteristics (ie age, BMI, Gleason score and clinical stage) in patients scheduled to undergo robot assisted laparoscopic radical prostatectomy were prospectively entered into a database. PPAT samples were procured during robot assisted laparoscopic radical prostatectomy according to a previously described technique25 and processed within 2 hours under a sterile laminar flow hood, as previously described.26 Immediately upon arrival the tissue was transferred to a Petri dish containing 20 ml PBS and finely minced in 20 to 80 mg pieces using scissors. Tissue pieces were extensively washed with 200 ml PBS

PERIPROSTATIC ADIPOSE TISSUE AND PROSTATE CANCER

over a 70 ␮m pore size filter (BD™ Biosciences). Thereafter the tissue pieces were transferred to a 50 ml centrifuge tube containing 45 ml 37C PBS and gently shaken for 20 minutes. The tube contents were poured over the filter and the tissue pieces were washed with 100 ml PBS at 37C. Tissue pieces were transferred to a tube containing 50 ml PBS and centrifuged for 1 minute at 277 ⫻ gravity at room temperature to remove red blood cells and debris. Tissue was then removed from the tube and weighed. PPAT was placed in a Petri dish with 10 ml M199 culture medium (Invitrogen™) supplemented with 50 ␮g/ml gentamicin per gm tissue. Medium collected after 20 to 24 hours was aliquoted into 1 ml fractions and immediately analyzed or stored at ⫺80C.

Multiplex Screening and Analyte Analysis Multiplex analysis of PPAT-CM was compared to patient matched serum in 7 preparations using a LINCOplex®-29 kit to screen for increased levels of a panel of proteins, including IL-1␣, IL-1␤, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12(p40), IL12(p70), IL-13, IL-15, IL-17, IFN-inducible protein-10 (CXCL10), MCP-1 (CCL2), macrophage inflammatory protein-1␣ (CCL3), macrophage inflammatory protein-1␤ (CCL4), sCD40L, transforming growth factor-␣, TNF␣, VEGF, epidermal growth factor, eotaxin (CCL11), fractalkine (CX3CL1), G-CSF, granulocyte-macrophage colony-stimulating factor and IFN-␥. The kit was used in conjunction with xMAP®. IL-6 levels in serum and PPAT-CM were further analyzed by ELISA according to manufacturer instructions (BioLegend, San Diego, California) and correlated with clinical variables such as age, BMI and Gleason score in 45 patients.

Immunohistochemistry Immunohistochemistry was performed in PPAT to characterize inflammatory infiltrates using CD45 and CD68 markers as well as IL-6 staining in 39 preparations and correlated with Gleason score. IL-6 staining was evaluated in 3 high power fields and samples were scored as strongly, moderately or weakly positive, or negative. Inflammatory cell infiltrate was quantified by screening tissue under low power for the highest density of cells, and then selecting 10 random high power fields and counting the number of positively stained cells.

Sample Preparation and Reverse Phase Protein Microarray Snap frozen PPAT samples weighing approximately 30 mg were subjected to alternating hydrostatic pressure cycling based lysis using a Barocycler NEP 3299 (Pressure BioSciences, West Bridgewater, Massachusetts).9 Using this approach fat tissue is subjected to alternating cycles of high and ambient pressure in a pressure generating instrument, which essentially liquefies tissue specimens. PPAT samples were subjected to 10 rapid pressure cycles, each consisting of 20 seconds at 35,000 psi followed by 20 seconds at ambient pressure. Resultant PPAT sample lysates were subjected to reverse phase tissue lysate array analysis, as previously described in detail,9 with a set of 14 phospho specific antibodies against signaling proteins known to be involved in cell survival, mitogenesis and cytokine signaling, including phospho Akt [(S473) and

1623

(T308)], phospho extracellular regulated kinase (T42/44), phospho P70S6 (S371), phospho c-erbB3 (Y1289), phospho AMPK␣ (S485), phospho AMPK␤ (S108), phospho I␬B␣ (S32/36), phospho STAT3 (Y705 and S727), phospho insulin receptor substrate 1 protein (S612), phospho mTOR (S2448), phospho STAT1 (Y701) and phospho JAK1 (Y701). Each antibody underwent rigorous validation before use (single band on Western blot and peptide competition). Each array was scanned individually on a NovaRay® Laser Scanner and saved as 16-bit TIF images. To normalize the phospho end point signal a separate array was stained with SYPRO® Ruby Stain, a highly sensitive fluorescent stain that generates a signal that is linearly proportional to the amount of protein on each spot. TIF images of antibody stained slides and SYPRO Ruby stained slide images were analyzed with MicroVigene™, version 2.200 image analysis software and Microsoft® Excel® 2000 software. Images were imported into MicroVigene to perform spot finding, local background subtraction, replicate averaging and total protein normalization, producing a single value for each sample at each end point.

Statistical Methods All statistical analysis was 2-sided using InStat®, version 3.0b for Macintosh®. Specific methods for each experiment are described.

RESULTS We examined levels of a panel of cytokines and growth factors in patients with prostate cancer undergoing robot assisted radical prostatectomy. Serum was obtained approximately 4 weeks before and approximately 3 months after surgery. We measured 29 cytokines and growth factors simultaneously in the same samples using a multiplex assay. No significant differences were detected among levels when samples were separated according to several clinical parameters, including serum PSA, tumor stage and patient BMI. Levels of several cytokines were highest in patients with low grade tumors. In 18 patient matched samples of serum obtained preoperatively and postoperatively there was no difference in cytokine or growth factor levels between the groups (4 weeks before vs 3 months after surgery) and no consistent direction of the change (increase or decrease) after surgery. PPAT from a subset of 7 matched preoperative and post-prostatectomy serum specimens was used to generate CM in a 24-hour period. PPAT CM was analyzed using the same multiplex assay as for serum. Of 28 analytes 12 were significantly higher in serum than PPAT-CM, including epidermal growth factor, CCL11, GM-CSF, IFN-␥, IL-1␣, IL-12p70, IL-15, IL-17, IL-2, IL-4, transforming growth factor-␣ and VEGF, which were below the detection level in PPAT CM. Conversely G-CSF, IL-6, IL-8 and CCL2 (MCP-1) were significantly higher in PPAT

1624

PERIPROSTATIC ADIPOSE TISSUE AND PROSTATE CANCER

CM than in patient matched serum, suggesting that PPAT may contribute significant levels of these factors to the prostate microenvironment. Samples were grouped according to Gleason grade and any cases of Gleason sum greater than 7 or any component of pattern 5 were classified as high grade. Levels of G-CSF, IL-6 and IL-8 in PPAT CM correlated with increasing Gleason grade but levels in patient matched serum obtained before prostatectomy were similar or lower in patients with higher grade tumors (fig. 1). Levels of IL-6, IL-8 and G-CSF in PPAT CM highly correlated with each other (each Pearson correlation coefficient ⬎0.95, p ⬍0.001). Based on these results we further examined IL-6 levels in PPAT in a larger cohort of 47 patients. Levels of IL-6 in PPAT CM again were significantly higher in patients with higher grade tumors, consistent with our initial observations using the multiplex assay (fig. 2). Higher IL-6 levels were also found in patients with higher stage tumors (pT2 compared to pT3 or 4), although this did not quite attain statistical significance (Mann-Whitney test p ⫽ 0.0527). Levels of IL-6 in PPAT CM did not correlate with patient age, BMI, patient matched serum PSA or serum IL-6. Of the patients 33 had matched IL-6 levels from preoperative serum and PPAT CM. There was no correlation between IL-6 levels in PPAT CM and serum,

indicating that IL-6 secreted from periprostatic fat does not contribute significantly to circulating IL-6 levels. These results clearly show that differences exist among patients with prostate cancer with respect to PPAT secreted cytokine levels and these levels appear to correlate with tumor aggressiveness but not with serum levels. Immunohistochemical staining of fixed PPAT with anti-IL-6 antibody revealed significant reactivity on adipocytes, although staining intensity in different samples did not correlate with the level of IL-6 secreted into the medium. Strong staining was detected in 5 of 13 Gleason 3 ⫹ 3, 12 of 19 Gleason 3 ⫹ 4, 2 of 4 Gleason 4 ⫹ 3 and 0 of 3 Gleason 4 ⫹ 5 samples. Since inflammatory cells are known to secrete high IL-6 levels, immunohistochemistry for CD68 and CD45 was performed to detect macrophages and lymphocytes. Inflammatory cells were not found in substantial numbers even in samples with the highest IL-6 levels in CM. In addition, there was no correlation between the number of inflammatory cells and Gleason score or IL-6 levels in PPAT CM. We queried 52 PPAT lysates using reverse phase protein microarray technology with a total of 14 phospho specific antibodies to quantitatively assess the relative levels of phosphorylation of several signaling proteins in the Jak/Stat, Akt/mTOR and nu-

Figure 1. Seven patient matched samples of serum and PPAT from patients undergoing radical prostatectomy for different grades of prostate cancer were analyzed using multiplex assay for 29 cytokines, chemokines and growth factors. Samples were stratified by Gleason grade with tumors containing any Gleason pattern 5 considered more aggressive and combined with Gleason sum greater than 7. Pearson correlation coefficients and p values were determined and only those of IL-6 were statistically significant.

PERIPROSTATIC ADIPOSE TISSUE AND PROSTATE CANCER

PPAT IL-6

A

80

40000

60

30000

pg/ml

20000

40 20

10000

Gleason

5 >7

or an y

7

>7

or an y

7

5

0