Human Cancer Biology

Urine Analysis and Protein Networking Identify Met as a Marker ofMetastaticProstateCancer Andrea L. Russo,1, 2 KimberlyJedlicka,3 Meredith Wernick,1Debbie McNally,1Melissa Kirk,1Mary Sproull,1 Sharon Smith,1Uma Shankavaram,1Aradhana Kaushal,1William D. Figg,4 William Dahut,4 Deborah Citrin,1Donald P. Bottaro,3 Paul S. Albert,5 Philip J. Tofilon,6 and Kevin Camphausen1

Abstract Purpose: Metastatic prostate cancer is a major cause of death of men in the United States.Expres- sion of met, a receptor tyrosine kinase, has been associated with progression of prostate cancer. Experimental Design: To investigate met as a biomarker of disease progression, urinary met was evaluated via ELISA in men with localized (n = 75) and metastatic (n = 81) prostate cancer. Boxplot analysis was used to compare the distribution of met values between each group. We estimated a receiver operating characteristic curve and the associated area under the curve to summarize the diagnostic accuracy of met for distinguishing between localized and metastatic disease. Protein-protein interaction networking via yeast two-hybrid technology supplemented by Ingenuity Pathway Analysis and Human Interactome was used to elucidate proteins and pathways related to met that may contribute to progression of disease. Results:Met distribution was significantly different between the metastatic group and the group with localized prostate cancer and people with no evidence of cancer (P < 0.0001).The area un- der the curve for localized and metastatic disease was 0.90, with a 95% confidence interval of 0.84 to 0.95.Yeast two-hybrid technology, Ingenuity PathwayAnalysis, and Human Interactome identified 89 proteins that interact with met, of which 40 have previously been associated with metastatic prostate cancer. Conclusion: Urinary met may provide a noninvasive biomarker indicative of metastatic prostate cancer and may be a central regulator of multiple pathways involved in prostate cancer progression.

Prostate cancer (PCa) is the most common non–skin cancer in Furthermore, it has been reported that PSA values may not men and the second most common cause of cancer deaths of directly correlate and, in some cases, may be inversely men in the United States, with nearly all deaths secondary to correlated, with disease progression. This phenomenon may metastatic disease (1). In the last two decades, serum prostate- be related to high levels of PSA resulting from architectural specific antigen (PSA) levels have been used for screening for distortion and low levels due to the loss of the ability to secrete PCa, as a marker of recurrence after therapy, and as a marker of PSA by tumor cells (5, 6). Despite the widespread use of PSA, metastatic disease; however, the utility of this test for screening concerns about the utility of PSA as a stand-alone test for and prediction of treatment outcome remains controversial (2). monitoring disease progression have prompted investigators to One limitation of PSA measurement is the findingthat patients seek alternative markers of disease progression. with metastatic disease in some cases have undetectable PSA Met, a receptor tyrosine kinase, has received much attention levels (3). Conversely, patients with elevated PSA levels for its overexpression and/or mutation in a number of posttreatment may never develop metastatic disease (4). malignancies (7). Multiple lines of evidence point to the importance of met in PCa initiation and progression. In vitro studies have shown that met expression is inversely correlated Authors’ Affiliations: 1Radiation Oncology Branch, National Cancer Institute, with androgen receptor expression (8). In murine models, 2HHMI/NIH Research Scholars Program, Howard Hughes Medical Institute, NIH, met ectodomain sheddinghas been shown to correlate with 3 4 5 Urologic Oncology Branch, Medical Oncology Branch, Biometric Research tumor burden across many histologies including PCa, and as Branch National Cancer Institute, Bethesda, Maryland; and 6Drug Discovery Program, H. Lee Moffitt Cancer Center,Tampa, Florida tumor burden increases, there is an increase in urinary and Received 3/10/09; revised 4/8/09; accepted 4/9/09; published OnlineFirst 6/23/09. plasma met levels (9). In prostatectomy specimens, met Grant support: Intramural Research Program of the NIH, National Cancer Institute expression was shown to correlate with progressive disease and by the Howard Hughes Medical Institute through the Howard Hughes Medical (10). A subsequent study showed similar results reporting Institute-NIHResearch Scholars Program. The costs of publication of this article were defrayed in part by the payment of page uniform met positivity in prostate bone metastasis (11). charges. This article must therefore be hereby marked advertisement in accordance Naughton et al. (12) extended these results by showing with 18 U.S.C. Section 1734 solely to indicate this fact. hepatocyte growth factor (HGF; the ligand of met) to be Requests for reprints: Kevin Camphausen, Radiation Oncology Branch, National significantly elevated in the serum of metastatic PCa patients Cancer Institute, 10 Center Drive 3B42, Bethesda, MD. Phone: 301-496-5457; when compared with patients with localized cancer. Thus, both Fax: 301-480-5439; E-mail: [email protected]. F 2009 American Association for Cancer Research. experimental as well as clinical data have associated met activity doi:10.1158/1078-0432.CCR-09-0599 with PCa progression.

Clin Cancer Res 2009;15(13) July 1,2009 4292 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2009 American Association for Cancer Research. Identify Met as a Marker of Metastatic Prostate Cancer

patients will attenuate any differences when comparinglocalized and Translational Relevance metastatic disease groups; thus, a test of our primary comparison will be conservative. The clinical and pathologic characteristics of patients with Prostate cancer is the most common cancer in men. localized PCa are shown in Table 1. We subsequently analyzed 81 Although prostate-specific antigen has been used to patient’s samples that had been collected on Protocols 00-C-0083 monitor disease progression and for screening purposes, it and 04-C-0257, clinical trials usingchemotherapy in patients is not a good marker for tumor progression as the disease with radiographic evidence of metastases from PCa. Urine was stored at -20jC, then thawed, centrifuged at 3,000 rpm for 10 min at 4jC, and becomes hormone insensitive. In this study, we examined supernatant was used for assay. Urinary creatinine levels were obtained the urinary biomarker met as a possible markerof metastatic on the Bayer DCA 2000+ Analyzer (Bayer HealthCare) accordingto PCa. Our data shows that urinary met is a significant marker manufacturer’s protocol. for patients with metastatic disease. Using yeast hybrid Electrochemiluminescence immunoassays. Met in urine specimens protein-protein interaction studies in combination with a was measured, in triplicate, usingelectrochemiluminescent immuno- bioinformatics approach, we show additional proteins that assay (Meso Scale Discovery), after adjustingto pH 6.5 to 7.5 according are related to met and may be part of the conversion of to the protocol described by Athauda et al. (9). Assays were done prostate cancer form a local disease to a systemic one. blinded to study end point. Our study sets the foundation for additional studies of Statistics. All analyses were done usingSPLUS version 7.0 (Insight- biomarkers of metastatic prostate cancer. ful corporation). Boxplots were used to descriptively compare the distribution of met values across low-risk, intermediate-risk, high-risk, normal, and metastatic patients. A Kruskal-Wallace nonparametric ANOVA test was used to compare the distributions of the localized Met has not been evaluated as an independent predictor of disease group. A Wilcoxon rank-sum test was used to compare values disease progression in PCa patients. To investigate the potential of normal individuals to the localized disease group. We estimated of met as a minimally invasive biomarker of disease progres- the receiver operatingcharacteristic curve (ROC) for distinguishing sion, we have evaluated urinary met in patients with both between the localized and metastatic groups using an empirical ROC localized and metastatic PCa. Furthermore, we used met as a curve. Area under the ROC curve (AUC) was estimated usinga bait in a protein-protein interaction (PPi) study combined with trapezoidal rule. A 95% confidence interval for the estimated AUC was obtained with a bootstrap usingthe percentile method with 5,000 an informatics approach to elucidate the proteins and pathways bootstrap samples. that may be involved in the progression of PCa from a local to a PPis. Myriad National Cancer Institute ProNet7 Yeast Two-hybrid metastatic disease. The data presented indicate that PSA and technology was used to identify PPis with met. Interactions with met levels are inversely correlated in prostate tumor cell lines met were tested usingnine baits, which covered the lengthof the and that higher urinary met levels are found in patients with met protein, and probed into three activation domain libraries metastatic PCa. Moreover, the PPi-informatics approach iden- (see Supplementary Table S1). Additional PPis were identified through tified a number of proteins previously recognized as being Unified Human Interactome (UniHI),8 an integrated resource from involved in PCa, but more importantly revealed a number of 10 major interaction maps derived by computational and experimental interactions between molecules that had not been previously methods. Ingenuity Pathways Analysis (IPA) was used as a third reported in PCa metastases. database of interactions. Once met interactingproteins were identified, a systematic PubMed search was completed to distinguish which proteins have been implicated in metastatic PCa. REMARK criteria. We used the REMARK (13) criteria as a guideline Materials and Methods in conductingand reportingthis study. This includes marker stated (met), study objectives, prespecified hypotheses, patient characteristics Cell lines and culture. The LNCaP [lymph node metastasis, (+) (Table 1), biological material used (urine), methods of preservation and PSA], PC-3 [bone metastasis, (-) PSA], DU145 [brain metastasis, (-) storage, assay method used, quality control, blinded to study end point, PSA], and 22Rv1 [prostate, (+) PSA] cell lines (American Type Culture stated retrospective study with prospective collection, time period of Collection), were maintained in DMEM supplemented with 10% fetal collection, specified statistical methods, and flow of patient study. Sample size for this hypothesis was based on usingall available samples. bovine serum in 5% CO2/95% air at 37jC. In vitro met and PSA quantitation. Cells were grown to 60% The relation to standard prognostic variables, results interpreted in the confluence, media changed, and 12 h later, cells were collected, protein context of prespecified hypotheses, limitations of study and implications quantified (Bradford assay), and ELISAs (total met and PSA; R&D for future research are described in the ‘‘Discussion’’ section. This Systems) were carried out in duplicate (40 Ag) according to study does not specifically discuss all patient treatments received, as all manufacturer’s protocol. patient specimens from those with localized disease were obtained Urine collection and processing. Urine was collected prospectively on pretreatment. This study does not report univariate or multivariate clinical study (02C0064), approved by the Institutional Review Board analyses in relation to outcome as the study was not designed to assess of the National Cancer Institute, NIH, from 2002 to present. Specimens patient outcome, and only 3 of 71 patients with localized disease failed were collected from 75 patients with localized PCa (18, 32, and 25 with within the study period. low, intermediate and high risk, respectively) before receiving radiation therapy and 20 males without PCa. Low-risk PCa is defined as clinical stage of T1 to T2a, Gleason score of 2 to 6, and PSA of <10 ng/mL; Results intermediate risk is defined as clinical stage of T2b to T2c, Gleason score of 7, and PSA of 10 to 20 ng/mL; and high risk is defined as clinical Recent in vitro studies have shown an inverse correlation stage of T3a, Gleason score of 8 to 10, and PSA of >20 ng/mL. Three between met protein levels and androgen receptor expression patients from the intermediate-risk group and one patient from the low- risk group had specimens with nondetectable levels of met and were excluded from analysis (poor specimen). Thus, we analyzed met from 7 https://pronet.myriad.com/nciweb/ 71 patients with localized disease. Excludingthe data from these four 8 http://theoderich.fb3.mdc-berlin.de:8080/unihi/home/

www.aacrjournals.org 4293 Clin Cancer Res 2009;15(13) July 1, 2009 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2009 American Association for Cancer Research. Human Cancer Biology

Table 1. Clinical and pathologic characteristics of patients with localized PCa

Overall (n = 71) Low (n = 17) Intermediate (n = 29) High (n = 25) PSA* 7.9 (0, 149) 3.2 (0, 8.7) 4.0 (0, 12) 16.4 (0,149) Gleason 5-6 31% (22) 100% (17) 10% (3) 8% (2) 7 41% (29) 0% (0) 90% (26) 12% (3) 8-10 28% (20) 0% (0) 0% (0) 80% (20) Clinical stage T1 51% (36) 53% (9) 55% (16) 44% (11) T2a 27% (19) 41% (7) 31% (9) 12% (3) T2b 10% (7) 6% (1) 14% (4) 8% (2) T2c 1% (1) 0% (0) 0% (0) 4% (1) T3a 8% (6) 0% (0) 0% (0) 24% (6) TX 3% (2) 0% (0) 0% (0) 8% (2) Hormones At enrollment 20% (14) 0% (0) 21% (6) 32% (8) Previously 7% (5) 12% (2) 7% (2) 4% (1)

*PSA is mean of each group, and () are ranges. () for other parameters are frequencies.

(8). To expand these initial findings and to better understand with localized PCa, divided into their clinical risk categories; the relationship between PSA and met in the progression of low, intermediate, or high, metastatic PCa, and a group of PCa, we measured the level of met and PSA in a panel of normal volunteers. As shown in Fig. 2, met could be detected in PCa cell lines from both local and metastatic sites (14). The the urine of patients with both localized and metastatic PCa as cell lines derived from a lymph node (LNCaP) or a primary well as the urine from men without cancer. There was no prostate specimen (22Rv1) had the highest PSA levels (68 and significant difference in met levels among any subgroups of 9.8 ng/mL, respectively). PC3 and DU145, derived from patients with localized disease (P = 0.44, Kruskal-Wallace metastatic bone and brain deposits, respectively, showed nonparametric ANOVA test comparingthe three groupsof nondetectable values for PSA. Conversely, PC3 and DU145 localized disease). Furthermore, there was no significant exhibited the greatest level of total met (3.4 and 3.76 ng/mL, difference between individuals without PCa and those with respectively), whereas 22Rv1 and LNCaP produced less total localized disease (P = 0.17, Wilcoxon rank-sum test). However, met (2.1 and 1.17 ng/mL, respectively). Thus, as shown in there was a highly significant difference between the normal Fig. 1, the level of total met and PSA are inversely correlated. group and the metastatic group (P < 0.0001) and between the Cell lines derived from distant metastatic sites expressed the localized disease group and the metastatic group (P < 0.0001). highest levels of met. Further correlation of met and PSA in patients with early We have previously shown that met could be detected in the metastatic disease may be important in determiningif met is an urine of mice bearinghuman xenografttumors (9). To accurate marker of PCa progression. determine if soluble met could be measured in the urine of To further assess the performance of urinary met to patients with PCa, urine was analyzed for met from patients distinguish between men with localized disease and men with metastatic disease, an empirical ROC curve was computed (Fig. 3). The AUC was 0.90 with a 95% confidence interval ranging from 0.84 to 0.95. This interval was evaluated using a bootstrap usingthe percentile method with 5,000 bootstrap samples. The data presented above suggest that met may provide a biomarker indicative of metastatic PCa. As an initial step toward understandingthe biologyunderlyingthis putative relationship between met and the metastatic progression of PCa, PPi involvingmet were identified usinga laboratory- based approach and two bioinformatics tools. First, an experimental method usinga yeast two-hybrid system was used to probe for proteins that directly bind to met. Three human tumor libraries were probed usingoverlappingmet fragments as baits. Six direct met PPi, as well as nine Fig. 1. PSA and total met protein levels are inversely correlated in PCa cell lines. secondarily derived interactions were identified (Table 2). In LNCaP, 22Rv1, PC-3, and DU145 PCa cell lines were grown to 60% confluency. addition, the UniHI database, which contains data from Cells were given fresh medium and were harvested 12 h later. Protein (40 Ag) was multiple PPi studies as well as literature-derived, computer- quantified by ELISA. Protein concentration (ng/mL) for each cell line is represented at % expression compared with the cell line with the highest concentration curated interactions, was queried. The UniHI database (LNCaP for PSA, DU145 for met). contained 43 met interactingproteins, of which 8 overlapped

Clin Cancer Res 2009;15(13) July 1,2009 4294 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2009 American Association for Cancer Research. Identify Met as a Marker of Metastatic Prostate Cancer

Discussion

PSA testingin patients with PCa has been used clinically for almost 20 years. Although originally approved for use in monitoringdisease progressionafter therapy, PSA monitoring is now widely used in screeningfor PCa. Interestingly,PSA is neither a very sensitive nor specific marker of disease progression (15). Thus, much work is currently under way to evaluate alternative markers (16). One such protein is the met receptor, the expression of which has been correlated with metastatic PCa. In contrast to other oncogenic proteins associated with prostate tumor progression, met ectodomain is shed from tumor cells and ultimately appears in the urine (9), thus providingthe potential for a noninvasive assessment. The studies described here evaluate the urinary met levels as a biomarker for metastatic PCa. As shown in Fig. 2, met is measurable in the urine of patients with PCa. Importantly, patients with metastatic PCa had higher levels of met than those with localized Fig. 2. Urinary met levels in normal volunteers and patients with PCa. Urinary met disease. However, met was also detectable in the urine of values were measured by electrochemiluminescent immunoassay. Samples were normal volunteers at levels not significantly different from run in triplicate and normalized with urinary creatinine levels. Low (T1-T2a, Gleason of 2-6, or PSA of <10 ng/mL), intermediate (T2b-T2c, Gleason of 7, or PSA of patients with localized PCa. The small sample size for each 10-20 ng/mL), and high (T3a, Gleason of 8-10, or PSA of >20 ng/mL) are risk subgroup may have obscured a small elevation in patients classification groups as defined by National Comprehensive Cancer Network with localized PCa, a hypothesis which could be tested in a guidelines. For each patient group, the line in the middle of the box represents the median. The lower and the upper edges of the box are the 1stand 3rd quartile, larger prospective clinical trial. Another possible explanation respectively.The fences are drawn to the nearest value not exceeding 1.5 is biologically based. In the normal prostate, HGF is produced (interquartile range), and observations beyond the fences are denoted as lines and are considered outliers. P value of <0.0001between metastatic and localized groups by stromal cells and met is present at high levels in basal and metastatic and normal groups. and intermediate cells, together acting in a paracrine fashion (17). Normal luminal cells express met only 2.1% of the time; however, expression increases to 44.1% with either with the Myriad data. Finally, IPA was used to define proteins proliferation or inflammation, commonly seen in patients interactingwith met. IPA contains interaction data that is with benign prostatic hypertrophy. Thus, a number of literature-derived but human-curated; thus, it can differ from physiologic conditions can potentially influence urinary met UniHI data. The IPA study revealed 58 proteins that directly interact with met. Of the 58 IPA-derived proteins, 8 overlapped with those identified through the yeast two-hybrid system, 19 overlapped with those from the UniHI database, and 8 were identified in all three methods. Thus, application of these three methods led to the identification of a total of 89 unique proteins that can interact with met. As the three methods/databases are constructed with different types of data and curated usingvarious methods, we used the entire set of 89 proteins in our literature search through the PubMed database. We entered, ‘‘protein name or synonym and prostate, PCa and/or metastatic PCa’’ in the search field. This search indicated that 40 of the 89 proteins had been associated with metastatic PCa as listed in Supplementary Table S1. Of these 40 proteins, 30 have been shown to be up-regulated and 10 proteins have been shown to be down-regulated in metastatic PCa. Four proteins (CBL, GRB2, HGF, and PLCG1) were common to all three databases and 10 proteins were common to two databases [BAG1, CTNNB1, CTTN, epidermal growth factor receptor, FAS, PLXNB1, PTPN11, SHC1, SRC, and signal transducers and activators of transcription 3 (STAT3)]. Of the 40 proteins, 19 were previously derived from the laboratory studies and 21 of the proteins were reported to interact with met strictly from a literature curation (Fig. 4). These data suggest that met may be a central regulator of multiple pathways involved in the progression of PCa from a local to Fig. 3. ROC analysis for localized versus metastatic PCa patients. AUC = 0.90; a metastatic disease. 95% confidence interval (CI), 0.84-0.95.

www.aacrjournals.org 4295 Clin Cancer Res 2009;15(13) July 1, 2009 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2009 American Association for Cancer Research. Human Cancer Biology

to met overexpression (18). One possible consequence of Table 2. met interacting proteins as identified such met overexpression would be increased sheddingand through 3independent bioinformatic databases elevated levels selectively in the urine of patients with metastatic PCa. Myriad ProNet UniHI IPA The mechanism accountingfor the increased met levels in Experimental Experimental/ Literature the urine of patients with advanced PCa remains speculation. computational To gain a better understanding of the biology underlying the GRAP BAG1 BAG1 elevated met expression in metastatic PCa, we used both GRAP2 CASP3 CBL GRB2 CBL CBLB experimental and bioinformatic approaches to develop a met PIK3R3 CNR1 CD44 interactome (Fig. 4). Met interacts with proteins that mediate PLCG1 CTNNB1 CD82 numerous processes characteristic of a metastatic phenotype ZYX CTTN CRKL Literature DAPK3 CTNNB1 includinginhibition of apoptosis, loss of cell adhesion, and CBL DNAJA3 CTTN enhancement of cell motility and migration (19). HGF, the GLMN EGFR DSG1 high-affinity ligand of met, binds to and activates met GAB1 FAS EGFR triggering a broad spectrum of biological responses. Several HGF FGF7 EHF mABL1 GAB1 ELF3 proteins we identified are involved in the stabilization/ mGRB10 GLMN F2 bindingof HGF, with subsequent activation of met, such as mPIK3p55 GRB2 FAS CD44 variant 3, PLAU and heparin sulfate (20). Other mPIK3R1 HGF FOS RANBP9 HGFAC GAB1 molecules that we identified are involved in the downstream HGS GAB2 signaling after HGF binding including, GRB2, PI3K, PLCg, IL32 GLMN RAS, SHC1, SRC, and Stat3 (21, 22). SHC1 binds directly to INPP5D GRB2 the phosphorylated receptor tail of met and, in combination INPPL1 Heparan sulfate ITGB4 HGF with GRB2, links met activation and the Ras–mitogen- MST1 JUN activated protein kinase pathway, previously shown to be MUC20 JUP important for cellular proliferation and transformation (23). PCBD2 MBP PIK3R1 MITF PI3K has been shown to be important for cell motility, PLCg PLCG1 MST1R for branchingmorphogenesis, and STAT3 for epithelial PLXNB1 MYH4 tubulogenesis (24). In an Oncomine analysis of microarray PTPN11 NRP1 data from patients with localized PCa versus those with PTPRB PIK3R1 PTPRJ PIK3C2B metastatic cancer, both GRB2 and STAT3 were over expressed RANBP10 PIK3R2 in the metastatic cancers (P =3.8Â 10-19 and 1.9 Â 10-5, RANBP9 PLAU respectively).9 Furthermore, HGF (also known as Scatter SH3KBP1 PLAUR SHC1 PLC GAMMA factor) is known to induce migration of cells, while inhibiting SMC1L1 PLCG1 intercellular adhesion (25). As shown in Supplementary SNAPAP PLG Table S2, CD82, DSG1, INPPL1, JUP, PTPRF, VIL2, and SNX2 PLXNB1 SPSB1 PLXNB2 zyxin (ZYX) are related to cell adhesion and are all down- SRC PLXNB3 regulated. Activation of met is known to lead to a loss STAT3 PP2A of cell-to-cell contact. For example, intact CD82 has been PTPN11 USP4 shown to suppress integrin-induced invasion of PCa cells VAV1 PTPRF in vitro YWHAZ RANBP9 and CD82 loss may promote metastasis (26). Likewise, RAS ZYX is known to bind myopodin leadingto slower migration RASA1 of PCa cells and a reduced invasiveness; thus, a loss of SHC1 SP1 ZYX might enhance migration and invasion (27). Thus, it SP3 follows that either an increase in total HGF, an increase in SPSB1 HGF binding, or an overexpression of met can lead to an SPSB2 SPSB3 increased signaling and in turn to a more invasive and SPSB4 metastatic phenotype. SRC A number of studies have used immunohistochemical STAT3 analyses of tissue specimens to associate the overexpression TGFa TP53 of met with metastatic PCa. The data reported here are the VIL2 first to show a significant increase in met levels in urine in WT1 patients with metastatic PCa. The use of urine as a source for biomarker analysis has the benefits of its ease of acquisition, NOTE: Bolded proteins are those found in all three databases noninvasiveness, and simplicity of storage and processing. (Myriad, UniHI, and IPA). Italicized proteins are those found in both As noted above, there was no apparent difference between UniHI and IPA. cancer-free males and those with localized PCa with respect to urinary met levels, indicatingthat met is unlikely to be levels. However, in advanced PCa, the paracrine HGF/met activity is thought to switch to an autocrine pathway; thus, as HGF becomes overexpressed in androgen-independent cancer cells, it may lead to hyper-stimulatingmet leading 9 http://www.oncomine.org

Clin Cancer Res 2009;15(13) July 1,2009 4296 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2009 American Association for Cancer Research. Identify Met as a Marker of Metastatic Prostate Cancer

Fig. 4. PPis with met were identified using Myriad National Cancer Institute ProNet, UniHI, and IPA. Of the 89 interacting proteins, the 40 illustrated here were found to be related to metastatic PCa. Inner circle, those experimentally/computationally found to be involved in metastatic PCa; outer circle, literature derived. Red and tan, up-regulated; green and blue, down-regulated. Bold text, those identified experimentally using Myriad.

applicable in initial diagnosis. However, the results presented comparison of met with bone scans and other imaging here suggest that urinary met may serve as a marker for modalities to determine if met can accurately identify patients metastatic PCa and may be particularly useful as a marker with metastatic disease before havingclinical or radiological of recurrence after therapy, in situations where PSA fails to evidence of such disease. identify such patients. Future investigation should compare ROC curves for both met and PSA, and based on this Disclosure of Potential Conflicts of Interest preliminary data, we would expect the AUC to be significantly smaller for PSA. Furthermore, studies should also include No potential conflicts of interest were disclosed.

References 1. JemalA,SiegelR,WardE,MurrayT,XuJ,ThunMJ. structure, functions and potential for therapeutic inhi- tumor marker prognostic studies (REMARK). J Natl Cancer statistics, 2007. CA Cancer J Clin 2007;57: bition. Cancer Metastasis Rev 2003;22:309 ^ 25. Cancer Inst 2005;97:1180 ^ 4. 43^66. 8.Verras M, Lee J, Xue H, Li TH, Wang Y, Sun Z. The 14. Sobel RE, Sadar MD. Cell lines used in prostate 2. Bensalah K, Lotan Y, Karam JA, Shariat SF. New androgen receptor negatively regulates the expres- cancer research: a compendium of old and new circulating biomarkers for prostate cancer. Prostate sion of c-Met: implications for a novel mechanism lines-part 1. J Urol 2005;173:342 ^ 59. Cancer Prostatic Dis 2007. of prostate cancer progression. Cancer Res 2007; 15. CatalonaWJ,RichieJP,AhmannFR,etal.Com- 3. Leibovici D, Spiess PE, Agarwal PK, et al. Prostate 67:967 ^ 75. parison of digital rectal examination and serum cancer progression in the presence of undetectable 9. Athauda G, Giubellino A, Coleman JA, et al. c-Met prostate specific antigen in the early detection of or low serum prostate-specific antigen level. Cancer ectodomain shedding rate correlates with malignant prostate cancer: results of a multicenter clinical trial 2007;109:198 ^ 204. potential. Clin Cancer Res 2006;12:4154 ^ 62. of 6,630 men. J Urol 1994;151:1283 ^ 90. 4. Fall K, Garmo H, Andren O, et al. Prostate-specific 10. Pisters LL, Troncoso P, Zhau HE, Li W, von 16. Laxman B, Morris DS,YuJ, et al. A first-generation antigen levels as a predictor of lethal prostate cancer. Eschenbach AC, Chung LW. c-met proto-oncogene multiplex biomarker analysis of urine for the early de- J Natl Cancer Inst 2007;99:526^ 32. expression in benign and malignant human prostate tection of prostate cancer. Cancer Res 2008;68: 5. Sandblom G, Ladjevardi S, Garmo H, Varenhorst tissues. J Urol 1995;154:293^ 8. 645^9. E. The impact of prostate-specific antigen level at 11. Knudsen BS, Gmyrek GA, Inra J, et al. High expres- 17. Nakashiro K, Hara S, Shinohara Y, et al. Phenotypic diagnosis on the relative survival of 28,531 men sion of the Met receptor in prostate cancer metastasis switch from paracrine to autocrine role of hepatocyte with localized carcinoma of the prostate. Cancer to bone. Urology 2002;60:1113^ 7. growth factor in an androgen-independent human 2008;112:813^ 9. 12. Naughton M, Picus J, Zhu X, Catalona WJ,Vollmer prostatic carcinoma cell line, CWR22R. Am J Pathol 6. Aihara M, Lebovitz RM, Wheeler TM, Kinner BM, RT, Humphrey PA. Scatter factor-hepatocyte growth 2004;165:533^40. Ohori M, Scardino PT. Prostate specific antigen and factor elevation in the serum of patients with prostate 18. Nakashiro K, Hayashi Y, Oyasu R. Immunohisto- gleason grade: an immunohistochemical study of cancer. J Urol 2001;165:1325^8. chemical expression of hepatocyte growth factor and prostate cancer. J Urol 1994;151:1558 ^ 64. 13 . McShane LM, Altman DG, Sauerbrei W, Taube SE, c-Met/HGF receptor in benign and malignant human 7. Ma PC, Maulik G, Christensen J, Salgia R. c-Met: Gion M, Clark GM. Reporting recommendations for prostate tissue. Oncol Rep 2003;10:1149 ^ 53.

www.aacrjournals.org 4297 Clin Cancer Res 2009;15(13) July 1, 2009 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2009 American Association for Cancer Research. Human Cancer Biology

19. Birchmeier C, Birchmeier W, Gherardi E, Vande The role of SF/HGF and c-Met in the develop- induction of prostate-specific antigen gene is re- Woude GF. Met, metastasis, motility and more. Nat ment of skeletal muscle. Development 1999;126: pressed by protein-protein interaction between the Rev 2003;4:915 ^ 25. 1621 ^ 9. androgen receptor and AP-1/c-Jun in the human pros- 20. van derVoort R,TaherTE,WielengaVJ, et al. Heparan 23. Ponzetto C, Zhen Z, Audero E, et al. Specific tate cancer cell line LNCaP. J Biol Chem 1997;272: sulfate-modified CD44 promotes hepatocyte growth uncoupling of GRB2 from the Met receptor. Differen- 174 8 5 ^ 9 4 . factor/scatter factor-induced signal transduction tial effects on transformation and motility. JBiol Chem 26. Sridhar SC, Miranti CK. Tetraspanin KAI1/CD82 through the receptor tyrosine kinase c-Met. J Biol 1996;271:14119 ^ 23. suppresses invasion by inhibiting integrin-depen- Chem 1999;274:6499^506. 24. Boccaccio C, Ando M, Tamagnone L, et al. Induc- dent crosstalk with c-Met receptor and Src 21. Liu X,Yao W, Newton RC, Scherle PA. Targeting the tion of epithelial tubules by growth factor HGF kinases. Oncogene 2006;25:2367 ^ 78. c-MET signaling pathway for cancer therapy. Expert depends on the STAT pathway. Nature 1998;391: 27. YuYP, Luo JH. Myopodin-mediated suppression of Opin Invest Drugs 2008;17:997 ^ 1011. 285 ^8. prostate cancer cell migration involves interaction with 22. Dietrich S, Abou-Rebyeh F, Brohmann H, et al. 25. Sato N, Sadar MD, Bruchovsky N, et al. Androgenic zyxin. Cancer Res 2006;66:7414^ 9.

Clin Cancer Res 2009;15(13) July 1,2009 4298 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2009 American Association for Cancer Research. Urine Analysis and Protein Networking Identify Met as a Marker of Metastatic Prostate Cancer

Andrea L. Russo, Kimberly Jedlicka, Meredith Wernick, et al.

Clin Cancer Res 2009;15:4292-4298.

Updated version Access the most recent version of this article at: http://clincancerres.aacrjournals.org/content/15/13/4292

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2021/03/12/1078-0432.CCR-09-0599.DC1

Cited articles This article cites 26 articles, 8 of which you can access for free at: http://clincancerres.aacrjournals.org/content/15/13/4292.full#ref-list-1

Citing articles This article has been cited by 4 HighWire-hosted articles. Access the articles at: http://clincancerres.aacrjournals.org/content/15/13/4292.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/15/13/4292. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.

Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2009 American Association for Cancer Research.