Identification of EpCAM as a Molecular Target of Prostate Cancer Stroma
2009; Elsevier BV; Volume: 175; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2009.090013
ISSN1525-2191
AutoresSumana Mukherjee, Annely M. Richardson, Jaime Rodriguez‐Canales, Kris Ylaya, Heidi S. Erickson, Audrey Player, Ernest S. Kawasaki, Peter A. Pinto, Peter L. Choyke, Maria J. Merino, Paul S. Albert, Rodrigo Chuaqui, Michael R. Emmert‐Buck,
Tópico(s)Heat shock proteins research
ResumoTo delineate the molecular changes that occur in the tumor microenvironment, we previously performed global transcript analysis of human prostate cancer specimens using tissue microdissection and expression microarrays. Epithelial and stromal compartments were individually studied in both tumor and normal fields. Tumor-associated stroma showed a distinctly different expression pattern compared with normal stroma, having 44 differentially expressed transcripts, the majority of which were up-regulated. In the present study, one of the up-regulated transcripts, epithelial cell adhesion activating molecule, was further evaluated at the protein level in 20 prostate cancer cases using immunohistochemistry and a histomathematical analysis strategy. The epithelial cell adhesion activating molecule showed a 76-fold expression increase in the tumor-associated stroma, as compared with matched normal stroma. Moreover, Gleason 4 or 5 tumor stroma was increased 170-fold relative to matched normal stroma, whereas the Gleason 3 tumor area showed only a 36-fold increase, indicating a positive correlation with Gleason tumor grade. Since the stromal compartment may be particularly accessible to vascular-delivered agents, epithelial cell adhesion activating molecule could become a valuable molecular target for imaging or treatment of prostate cancer. To delineate the molecular changes that occur in the tumor microenvironment, we previously performed global transcript analysis of human prostate cancer specimens using tissue microdissection and expression microarrays. Epithelial and stromal compartments were individually studied in both tumor and normal fields. Tumor-associated stroma showed a distinctly different expression pattern compared with normal stroma, having 44 differentially expressed transcripts, the majority of which were up-regulated. In the present study, one of the up-regulated transcripts, epithelial cell adhesion activating molecule, was further evaluated at the protein level in 20 prostate cancer cases using immunohistochemistry and a histomathematical analysis strategy. The epithelial cell adhesion activating molecule showed a 76-fold expression increase in the tumor-associated stroma, as compared with matched normal stroma. Moreover, Gleason 4 or 5 tumor stroma was increased 170-fold relative to matched normal stroma, whereas the Gleason 3 tumor area showed only a 36-fold increase, indicating a positive correlation with Gleason tumor grade. Since the stromal compartment may be particularly accessible to vascular-delivered agents, epithelial cell adhesion activating molecule could become a valuable molecular target for imaging or treatment of prostate cancer. The stromal compartment in tissues is often considered a passive mechanical support for epithelial cells; however, recent evidence indicates that the stroma plays a critical role in many important biological processes.1Cunha GR Hayward SW Dahiya R Foster BA Smooth muscle-epithelial interactions in normal and neoplastic prostatic development.Acta Anat (Basel). 1996; 155: 63-72Crossref PubMed Scopus (157) Google Scholar, 2Podlasek CA Barnett DH Clemens JQ Bak PM Bushman W Prostate development requires Sonic hedgehog expressed by the urogenital sinus epithelium.Dev Biol. 1999; 209: 28-39Crossref PubMed Scopus (188) Google Scholar, 3Johnson RL Tabin CJ Molecular models for vetebrate limb development.Cell. 1997; 90: 979-990Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar, 4Mericskay M Kitajewski J Sassoon D Wnt5a is required for proper epithelial-mesenchymal interactions in the uterus.Development. 2004; 131: 2061-2072Crossref PubMed Scopus (200) Google Scholar, 5Cunha GR Cooke PS Kurita T Role of stromal-epithelial interactions in hormonal responses.Arch HistolCytol. 2004; 67: 417-434Crossref PubMed Scopus (237) Google Scholar, 6Tlsty TD Hein PW Know thy neighbor: stromal cells can contribute oncogenic signals.Curr Opin Genet Dev. 2001; 11: 54-59Crossref PubMed Scopus (324) Google Scholar, 7Bergers G Coussens LM Extrinsic regulators of epithelial tumor progression: metalloproteinases.Curr Opin Genet Dev. 2000; 10: 120-127Crossref PubMed Scopus (122) Google Scholar, 8Kiaris H Chatzistamou I Kalafoutis Ch Koutselini H Piperi Ch Kalofoutis A Tumor-stroma interactions in carcinogenesis: basic aspects and perspectives.Mol Cell Biochem. 2004; 261: 117-122Crossref PubMed Scopus (36) Google Scholar, 9Bhowmick NA Chytil A Plieth D Gorska AE Dumont N Shappell S Washington MK Neilson EG Moses HL TGF-beta signaling in fibroblasts modulates the oncogenic potential of adjacent epithelia.Science. 2004; 303: 848-851Crossref PubMed Scopus (1137) Google Scholar, 10Cunha GR Hayward SW Wang YZ Ricke WA Role of the stromal microenvironment in carcinogenesis of the prostate.Int J Cancer. 2003; 107: 1-10Crossref PubMed Scopus (316) Google Scholar, 11Tlsty TD Coussens LM Tumor stroma and regulation of cancer development.Annu Rev Pathol Mech Dis. 2006; 1: 119-150Crossref PubMed Scopus (807) Google Scholar For example, both in vivo and ex vivo studies have shown that dynamic epithelial-stroma interactions influence branching morphogenesis during glandular development, and affect angiogenesis during tissue specific differentiation.12Ghosh K Ingber D Micromechanical control of cell and tissue development: implications for tissue engineering.Adv Drug Delivery Rev. 2007; 59: 1306-1318Crossref PubMed Scopus (169) Google Scholar, 13Cunha GR Ricke W Thomson A Thomson A Marker PC Risbridger G Hayward SW Wang YZ Donjacour AA Kurita T Hormonal, cellular, and molecular regulation of normal and neoplastic prostatic development.J Steroid Biochem Mol Biol. 2004; 92: 221-236Crossref PubMed Scopus (244) Google Scholar Similarly, macrophage association with the developing mammary gland is critical during embryogenesis as evidenced by the fact that colony stimulating factor-1 or colony stimulating factor-1 receptor null mice (devoid of macrophage) have defective mammary glands.14Cunha GR Young P Hom YK Cooke PS Jaylor JA Lubahn DB Elucidation of a role for steroid hormone receptors in mammary gland growth and development.J Mammary Gland Biol Neoplasia. 1997; 2: 393-402Crossref PubMed Scopus (180) Google Scholar In neoplasia, several lines of evidence suggest that stromal abnormalities contribute to tumorigenesis. Genome-based studies indicate stromal cells are altered in some inherited cancer susceptibility syndromes,15Bissell MJ Radisky D Putting tumors in context.Nat Rev Cancer. 2001; 1: 46-54Crossref PubMed Scopus (1734) Google Scholar genomic rearrangements at several loci are observed in tumor-associated stromal cells,16Fukino K Shen L Patocs A Mutter G Eng C Genome instability within tumor stroma and clinicopathological characteristics of sporadic primary invasive breast carcinoma.JAMA. 2007; 297: 2103-2111Crossref PubMed Scopus (103) Google Scholar, 17Moinfar F Man YG Arnould L Bratthauer GL Ratschek M Tavassoli FA Concurrent and independent genetic alterations in the stromal and epithelial cells of mammary carcinoma: implications for tumorigenesis.Cancer Res. 2000; 60: 2562-2566PubMed Google Scholar and genetic alterations in the stroma may precede genotypic changes in epithelial tumors.16Fukino K Shen L Patocs A Mutter G Eng C Genome instability within tumor stroma and clinicopathological characteristics of sporadic primary invasive breast carcinoma.JAMA. 2007; 297: 2103-2111Crossref PubMed Scopus (103) Google Scholar, 17Moinfar F Man YG Arnould L Bratthauer GL Ratschek M Tavassoli FA Concurrent and independent genetic alterations in the stromal and epithelial cells of mammary carcinoma: implications for tumorigenesis.Cancer Res. 2000; 60: 2562-2566PubMed Google Scholar, 18Radisky D Hagios C Bissell MJ Tumors are unique organs defined by abnormal signaling and context.Sem Cancer Biol. 2001; 11: 87-95Crossref PubMed Scopus (150) Google Scholar Moreover, heritable genetic defects that affect the stroma have also been identified in juvenile polyposis and in syndromes associated with endometrial polyps.19Howe JR Roth S Ringold JC Summers RW Jarvinen HJ Sistonen P Tomlinson IP Houlston RS Bevan S Mitros FA Mutations in the SMAD4/DPC4 gene in juvenile polyposis.Science. 1998; 280: 1086-1088Crossref PubMed Scopus (760) Google Scholar, 20Schreibman IR Baker M Amos C McGarrity TJ The hamartomatous polyposis syndromes: a clinical and molecular review.Am J Gastroenterol. 2005; 100: 476-490Crossref PubMed Scopus (268) Google Scholar, 21Chow E Macrae F A review of juvenile polyposis syndrome.J Gastroenterol Hepatol. 2005; 20: 1634-1640Crossref PubMed Scopus (116) Google Scholar Gene expression changes in stromal cells, or expression alterations that affect stromal-epithelial interactions can also influence the development of invasive epithelial tumors, either positively or negatively.22Li H Fan X Houghton J Tumor microenvironment: the role of the tumor stroma in cancer.J Cell Biochem. 2007; 101: 805-815Crossref PubMed Scopus (462) Google Scholar, 23Elanbaas B Weinberg RA Heterotypic signaling between epithelial tumor cells and fibroblasts in carcinoma formation.Exp Cell Res. 2001; 264: 169-184Crossref PubMed Scopus (448) Google Scholar As an example, bone morphogenetic protein antagonist germline 1 is widely expressed by cancer-associated stromal cells and provides a favorable microenvironment for cell survival and expansion.24Sneddon JB Zhen HH Montgomery K Rijn M Tward AD West R Gladstone H Chang HH Morganroth GS Oro AE Brown PO Bone morphogenetic protein antagonist gremlin 1 is widely expressed by cancer-associated stromal cells and can promote tumor cell proliferation.Proc Natl Acad Sci USA. 2006; 103: 14842-14847Crossref PubMed Scopus (235) Google Scholar Alternatively, attenuation of β1-integrin (laminin receptor) in highly aggressive human breast cancer cells leads to reorganization of the cytoskeleton, redistribution of β-catenin and E-cadherin, formation of adherens junctions, and alteration in signaling pathways that result in a reversion of the aggressive phenotype.25Kenny PA Bissell MJ Tumor reversion: correction of malignant behaviour by microenvironmental cues.Int J Cancer. 2003; 107: 688-695Crossref PubMed Scopus (279) Google Scholar In addition to the influence of genomic status and gene expression levels, several experiments have shown that the physical presence of stromal cells, such as tumor-associated fibroblasts can directly influence the malignant progression of cancer. Human prostatic epithelial cells show dramatic changes both in histology and growth rate when grown with human fibroblast cells derived from prostatic carcinoma, and co-injection of fibroblasts with tumor epithelial cells into mice enhances tumor formation.26Olumi AF Grossfeld GD Hayward SW Carroll PR Tlsty TD Cunha GR Carcinoma-associated fibroblasts direct tumor progression of initiated human prostatic epithelium.Cancer Res. 1999; 59: 5002-5011PubMed Google Scholar, 27Camps JL Chang SM Hsu TC Freeman MR Hong SJ Zhau HE VonEschenbach AC Chung LW Fibroblast-mediated acceleration of human epithelial tumor growth in vivo.Proc Natl Acad Sci USA. 1990; 87: 75-79Crossref PubMed Scopus (375) Google Scholar Taken together, these genomic, gene expression, and cell-based observations suggest that alterations in the stroma can significantly affect cell proliferation and tumor development. To assess the molecular profile of the tumor-associated stroma in prostate tissues, microdissected epithelial and stromal cells from normal and tumor regions of human prostatectomy specimens were previously analyzed at the transcriptome level. Forty-four genes were differentially expressed in the tumor-associated stroma, including epithelial cell adhesion activating molecule (EpCAM), an epithelial glycoprotein.28Richarson A Woodson K Wang Y Rodriguez-Canales J Erickson HS Tangrea MA Novakovic K Gonzalez S Velasco A Kawasaki ES Emmert-Buck MR Chuaqui RF Player A Global expression analysis of prostate cancer-associated stroma and epithelia.J Mol Diagn. 2007; 16: 189-197Crossref Scopus (42) Google Scholar In the present study we analyzed the expression of EpCAM at the protein level in prostate cancer patients using immunohistochemical staining of prostatectomy sections, coupled with a histomathematical analysis that allowed us to quantitatively measure protein amounts in the tumor microenvironment. Prostatectomy cases were obtained from the National Institutes of Health and the National Naval Medical Center under an institutional review board-approved protocol. A total of 20 cases were studied, including nine flash frozen tissues embedded in optimal cutting compound medium, and 11 prostate cases that were ethanol fixed and paraffin embedded as described previously.29Gillespie JW Best CJ Bichsel VE Cole KA Greenhut SF Hewitt SM Ahram M Evaluation of non-formalin tissue fixation for molecular profiling studies.Am J Pathol. 2002; 160: 449-457Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar Two pathologists (R.F.C. and J.R.-C.) evaluated the sections and concurred on Gleason scores. All cases contained localized prostate cancer. Immunohistochemical staining of ethanol-fixed and paraffin-embedded sections for EpCAM expression was performed using a standard immunohistochemistry (IHC) protocol. Each 5-um thick section was heated at 60°C for 1 hour and then deparaffinized, rehydrated, and incubated with 0.3% hydrogen peroxide for 30 minutes at room temperature to block endogenous peroxidase activity. Frozen sections were incubated in 70% ethanol for 10 minutes at room temperature and blocked with hydrogen peroxide for 30 minutes before IHC. After rinsing in 0.1 M/L PBS (pH 7.4), sections were incubated for 1 hour at room temperature with EpCAM primary mouse monoclonal antibody (Abcam, cat. #11294) diluted 1:200 with antibody diluent (Zymed). Sections were incubated for 1 hour with goat anti-mouse secondary antibody conjugated to peroxidase (Dako). The chromogen 3, 3′-diaminobenzidine tetrahydrochloride was applied as a 0.02% solution containing 0.005% hydrogen peroxide in 50 mmol/L ammonium acetate-citrate acid buffer (pH 6.0). The sections were lightly counterstained in Mayer's hematoxylin and mounted. Negative controls were established by replacing the primary antibody with antibody diluent and no detectable staining was evident in these sections. Normal epithelium in the tissue was used as a positive control for EpCAM staining. Positive reactions (ie, positively stained cells) were identified by the presence of a brown precipitate. Negative reactions (ie, negative cells) were identified by the absence of a brown precipitate and only blue counterstain. IHC on the same sections was performed for pan-cytokeratin AE1/AE3 (Invitrogen, cat. # 18–0132) at a dilution of 1:50 to differentiate epithelial from stromal cells. Additionally, two ethanol-fixed, paraffin-embedded sections were used for IHC staining of CD31 (Dako, cat. #) at a dilution of 1:50 to label endothelial cells. Two pathologists (R.F.C. and J.R.-C.) analyzed the IHC-stained tissue sections and photographed representative regions with an Olympus microscope and a charged-couple device camera using ×20 magnification and a resolution of 2080 × 1542 pixels (CCD color bayer mosaic; Q-color-3, Olympus America Inc., Melville, NY). In each case, four images were taken per distinct Gleason focus, as well as one arbitrarily selected field containing only normal cells. The immunostaining of all of the images was evaluated by three authors (S.M., R.F.C., and J.R.-C.) and EpCAM expression in the stroma was analyzed initially using a semiquantitative method, followed later by a more quantitative histomathematical analysis to measure the fold-change in different tumor grades compared with normal. For semiquantitative analysis, stromal staining was visually classified into four categories: 1+ (10% to 20% tumor stroma stained), 2+ (20% to 50%), 3+ (50% to 90%), 4+ (more than 90%). For quantitative analysis, all images were initially scanned and analyzed using the ImagePro Analysis System (ImagePro 4.5; Cybernetics, Chevy Chase, MD) as described previously.30Gannot G Gillespie JW Chuaqui FC Tangrea MA Linehan MW Emmert-Buck MR Histomathematical analysis of clinical specimens: challenges and progress.J Histochem Cytochem. 2005; 53: 177-185Crossref PubMed Scopus (4) Google Scholar Images were examined using the ACDSee program (ACD Systems of America; Miami, Fl) and the IHC-positive staining was evaluated according to the most intensely stained and the least intensely stained image. The data were collected based on the brown staining in the intensely stained image (ie, positively stained cells) and for the blue staining in the least intensely stained case (ie, negative cells). The subsequent epithelial measurements were based on positive versus negative cell count; whereas, the stromal measurements were based on topographical regions that were positive or negative, thereby including both cell- and matrix-associated EpCAM staining. The ImagePro watershed separation was used for the image analysis. Each measurement was exported to an MS Excel (Microsoft Excel 2000; Seattle, WA) spreadsheet and the mean staining was calculated from the four images analyzed for each set. Tumor foci that showed different Gleason grades within each tissue were identified and photographed. One distinct tumor was identified in 17 cases and two tumors were identified in three cases. Therefore, a total of 23 tumors were analyzed (Table 1).Table 1Summary of EpCAM Protein Levels in Normal and Tumor Regions of the ProstateCell typeStromal EpCAMNormal, n = 20Stain-0 ( 90%) = 3 Open table in a new tab Four multiple subregion staining values were averaged across each normal region and each tumor region. To obtain a single average measurement for both normal and tumor staining, we also averaged staining values across multiple tumors on the same subject. A Wilcoxon signed rank test was used to determine whether the staining was different between tumor and matched normal tissue. We examined whether the difference between tumor and matched normal tissue varied by Gleason score using a Wilcoxon rank sum test. In this latter analysis, we dichotomized Gleason score and compared changes between tumor and matched normal tissue for tumors with Gleason 3 versus Gleason 4/5. All tests were two-sided and a P value <0.05 was considered statistically significant. EpCAM protein levels in twenty human prostate specimens containing 23 independent tumors were studied. In each case, IHC measurements of EpCAM were taken from whole-mount sections containing normal stroma, normal epithelium, tumor stroma, and tumor epithelium, and analyzed using both a semiquantitative and a quantitative histomathematical approach (Figure 1). To ensure the results were not significantly affected by tissue processing, both frozen samples and ethanol-fixed, paraffin-embedded specimens were included in the study and similar results were obtained for both. IHC for cytokeratin was also performed as a control to identify possible tumor cells in the stroma and to distinguish their staining from true stromal staining (Figure 2A–F, Figure 3A–F).Figure 2Serial sections of two tissue specimens from a patient with prostate cancer. The first sample contains normal epithelium and stroma (A–C), and the second sample contains normal epithelium, stroma, and tumor cells (D–F). A, D: H&E-staining. B, E: Cytokeratin staining. C, F: EpCAM staining. Strong EpCAM staining is seen in both normal and tumor epithelium, but is selectively observed in the stroma associated with tumor (F, bottom half).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Serial sections of a tissue sample from a second patient with prostate cancer. The sample contains normal epithelium, stroma, and tumor cells. Images were taken at ×10 magnification (A–C) and ×40 magnification (D–F). A, D: H&E staining. B, E: Cytokeratin staining. C, F: EpCAM staining. EpCAM staining is seen in both normal and tumor epithelium, but is selectively observed in the stroma associated with tumor (C and arrows in F).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Consistent and reproducible EpCAM staining was observed in all 20 cases. Epithelium showed a membranous and diffuse pattern both in normal and tumor cells with no difference in staining intensity (data not shown). No stromal staining was observed in histological fields containing only normal cells (Figure 2C). However, in the tumor regions strong staining was present in the stroma (Figure 2, Figure 3, Figure 4; Table 1; Table 2).Table 2EpCAM IHC Staining in the Tumor Microenvironment (Epithelial and Stromal) and in the Tumor-Associated StromaTotal staining (epithelial and stromal)Tumor stromal stainingSampleCaseStaining valueAverageSDStaining valueAverageSDN-10.3504750.51770.1144940.00004680.0003180.00037351N-20.5388640.000135N-30.6009260.0002234N-40.58085040.0008681-(Gl: 3)T-10.402380.42300.0509420.0427440.0223910.013735T-20.49850.12452T-30.38650.018099T-40.40490.0162401-(Gl: 4)T-10.54996790.49950.062860.0459780.03774350.0076948T-20.455840.035979T-30.4355140.027935T-40.5570060.040972N-10.33037630.3168.0826810.000440.00019030.0001707N-20.38354670.0001427N-30.35628090.000124N-40.19717480.00005452-(Gl: 3)T-10.2196830.23140.0531220.00947460.014410.005023T-20.1646210.008177T-30.2904880.03022T-40.2508340.0097833N-10.3891290.29540.078540.00006440.00012890.0004421N-20.2439240.000988N-30.3300380.00031N-40.2187580.0000413-(Gl: 4)T-10.3077580.214960.064130.01974050.01215360.0056938T-20.1633820.008668T-30.2048160.0132022T-40.1839270.007014N-10.1286350.08230.0388840.00007550.00006840.0000169N-20.09760.0000859N-30.0399080.0000657N-40.0633810.00004624-(Gl: 4)T-10.3010100.235480.0691580.0078960.00733790.0018856T-20.2894570.00599T-30.1768230.00976T-40.174650.0057025N-10.3733120.35070.1677720.00025380.00020870.000162N-20.117070.0001275N-30.515680.0000404N-40.3968510.00041295-(Gl: 4)T-10.482440.4839.042920.0474190.026750.0150277T-20.5261830.01176T-30.501610.02608T-40.4253930.0217366N-10.241160.14440.098520.00052910.0002220.0002175N-20.213200.0002196N-30.087250.000094N-40.036040.0000456-(Gl: 3)T-10.342580.37660.029020.021430.022220.0062448T-20.3719760.015980T-30.4132750.030870T-40.3785580.0206037N-10.219490.14150.059870.00039520.00018530.0001478N-20.0736530.0001718N-30.133500.0001210N-40.139530.00005427-(Gl: 4)T-10.067640.15100.1005820.00133180.00570.0071163T-20.140470.0032546T-30.295210.0163074T-40.101030.0019128N-10.140680.13710.0366950.00072680.00028820.0002953N-20.117070.0002011N-30.18720.0001087N-40.10370.00011638-(Gl: 3)T-10.424530.44210.018160.00706050.0208780.0193542T-20.466030.0231231T-30.445720.0332446T-40.432340.0200845(table continues)8-(Gl: 4)T-10.936210.924850.129720.0354530.0310750.0041912T-20.676080.029356T-30.6491860.033150T-40.771040.0025949N-10.5985480.45630.119800.00343800.00408350.0008508N-20.403250.005336N-30.322230.003798N-40.5014480.00376129-(Gl: 3)T-10.530860.58920.046500.0455680.05762750.01018T-20.574130.052780T-30.634540.0661355T-40.6174750.06596910N-10.044150.05380.0491760.00004960.00028090.0004707N-20.007800.0000232N-30.039930.0000642N-40.123460.000986510-(Gl: 4)T-10.27276560.20720.0806860.0172800.0103690.0055567T-20.2701180.009389T-30.1040560.003756T-40.1820090.010805610-(Gl: 5)T-10.444110.51560.139030.06556670.04860660.0249433T-20.728430.083281T-30.6453290.025990T-40.46330.07222211N-10.0597440.08960.0372920.00007550.00006180.0000291N-20.14413450.0000859N-30.077390.0000657N-40.0771280.000019911-(Gl: 3)T-10.14111650.14590.0655390.00299260.00453570.0029695T-20.2142030.0059906T-30.17002130.0078955T-40.058630.001263912N-10.191470.18280.0322410.00008640.00012410.0000377N-20.22220.0000991N-30.145670.0001419N-40.17220.000168912-(Gl: 4)T-10.50290.46330.0384810.04741950.032490.0135662T-20.46810.0360844T-30.41060.0317362T-40.47190.0147213N-10.23140.20260.0382130.00011190.00009950.0000212N-20.146290.0000803N-30.215370.0000827N-40.217340.00012313-(Gl: 3)T-10.35130.36530.0565920.01647330.0171220.0072624T-20.280070.0109118T-30.32850.0274695T-40.41630.013633414N-10.0693220.08550.0368030.00028640.000120.0001169N-20.104360.0000821N-30.1256440.000099N-40.042670.00001214-(Gl: 5)T-10.30520.47670.303580.0299260.045810.028999T-20.391540.059906T-30.338900.078955T-40.94820.01461215N-10.29040.25710.0623750.00025380.0001040.0001151N-20.29250.0000296N-30.28170.0000275N-40.16380.000014915-(Gl: 3)T-10.75600.72380.0320560.03120580.0354530.0064386T-20.70590.037982T-30.68850.0433648T-40.74570.029259416N-10.27630.21820.0925440.00011190.0000810.0000443N-20.31750.0000803N-30.13360.0000082N-40.14540.0000488(table continues)16-(Gl: 4)T-10.84740.59780.174480.03120580.0409720.01246T-20.55970.0533648T-30.44100.0379818T-40.54310.041335417N-10.09570.10600.0099160.00007550.0000880.0000286N-20.11360.0000859N-30.11340.0001275N-40.09700.000062817-(Gl: 4)T-10.43190.48210.1151420.0360720.025810.0116898T-20.37510.0203954T-30.64270.034866T-40.47870.011906218N-10.23190.21750.0568390.00025380.00018930.000129N-20.17840.0001275N-30.16800.0000404N-40.29170.0003318-(Gl: 5)T-10.37800.47510.0750660.03438080.037960.0321204T-20.55630.0379818T-30.46360.078955T-40.50250.0005219N-10.12860.09210.0313240.00007550.0000960.0000413N-20.05720.0000859N-30.10530.0000657N-40.07730.000156719-(Gl: 4)T-10.42080.33280.0738130.0493240.028320.0141159T-20.25120.0214745T-30.29860.0234028T-40.36060.019078520N-10.09430.11440.0355230.00028640.000120.0001137N-20.14570.0000991N-30.14300.0000419N-40.07460.000052520-(Gl: 4)T-10.23330.38330.1622320.02340280.02640.0297138T-20.28330.0067544T-30.42110.0693240T-40.59550.0061186 Open table in a new tab An initial semiquantitative analysis of EpCAM levels in the tumor-associated stroma showed: Four+ expression in 3/23 tumors (13%). Twelve out of the 23 samples were scored as 3+ (52%). A staining score of 2+ was observed in 6/23 (26%) tumors, and weak staining was present in 2/23 (9%) (Table 1). With respect to topography, the distribution of stromal staining was maximal near the tumor epithelium and gradually decreased as the distance from tumor cells increased. The staining was next measured using a quantitative image scoring system (Tables 2 and 3). Overall, there was a statistically significant (P < 0.001) two-fold increase in EpCAM staining in the tumor microenvironment compared with normal regions when both epithelial and stromal components were included in the analysis (Table 3). However, when only stromal staining was analyzed, there was a 76-fold increase in EpCAM staining in the tumor stroma (0.0266) compared with normal (0.000352) (P < 0.001).Table 3Summary of EpCAM Levels in Normal and Tumor AreasAll cells (epithelial + stromal)Stromal cellsNormalTumorNormalTumor0.203.4120.000352.0266 Open table in a new tab The EpCAM stromal staining was most pronounced in histological fields that contained high-grade Gleason foci as is shown in Figure 4, A–F. Tumors were segregated according to their total Gleason grade sum into high-grade (Gleason grade 4 or 5) and moderate/low-grade (Gleason grade 3 or less) and analyzed for stromal staining as well as total (epithelium + stroma) staining (Table 4). Stromal staining for Gleason 4/5 foci was increased 170-fold relative to matched normal stroma, whereas the lower-grade Gleason tumors showed only a 36-fold increase in staining compared with matched normal stroma. Although these differences were not statistically significant (P = 0.38), the direction of the effect suggests that EpCAM protein levels in the tumor stroma increases with Gleason grade. The lack of statistical significance may be due to the small sample size.Table 4EpCAM Levels Correlated with Gleason GradeGleason gradeNumber of samplesAll cells (epithelium+stroma)StromaTumor fold increase over matched normalP valueTumor fold increase over matched normalP value381.60.08360.0084/5152.30.0011700.001 Open table in a new tab We performed endothelial cell IHC labeling with anti-CD31 antibody to analyze the distribution of vessels in both the normal and tumor areas with special attention on the relationship of neovessels and EpCAM staining in tumor stroma. Most of the small vessels in normal areas were distributed around glandular epithelium or lobules, as expected. However, rich neovascularization was seen in tumor areas, with little or no organization and irregularly shaped vessels throughout the tumor stroma (Figure 5, A–F). In the present study, we show that EpCAM protein is up-regulated in tumor-associated stroma of the prostate. All of the primary cases analyzed showed an increase of EpCAM in the tumor areas compared with matched normal regions from the same patients. These results are consistent with our previous mRNA data, where EpCAM was one of the highest up-regulated transcripts in tumor versus normal stroma.28Richarson A Woodson K Wang Y Rodriguez-Canales J Erickson HS Tangrea MA Novakovic K Gonzalez S Velasco A Kawasaki ES Emmert-Buck MR Chuaqui RF Player A Global expression analysis of prostate cancer-associated stroma and epithelia.J Mol Diagn. 2007; 16: 189-197Crossref Scopus (42) Google Scholar The precise role of EpCAM in tumorigenesis is not currently known. EpCAM is an adhesion molecule usually expressed on the basolateral membrane of epithelial cells and has been found to be overexpressed on carcinomas and cancer-initiating cells.31Went P Dirnhofer S Schopf D Moch H Spizzo G Expression and prognostic significance of EpCAM.J Cancer Molecules. 2008; 3: 169-174Google Scholar Recently, Maetzel et al32Maetzel D Denzel S Mack B Canis M Went P Benk M Kieu C Papior P Baeuerle PA Munz M Gires O Nuclear signaling by tumor-associated antigen EpCAM.Nature Cell Biology. 2009; 11: 162-171Crossref PubMed Scopus (544) Google Scholar identified EpCAM as a potent mitogenic signal transducer, promoting cell cycling and enhancing proliferation. Signaling by EpCAM requires regulated intramembrane proteolysis catalyzed by two proteases, TACE and PS-2, thus EpCAM growth-promoting effects may potentially be inhibited by pharmacological inhibition of TACE and/or PS-2. Interestingly, EpCAM seems to have different regulatory pathways in different tumor types. It can be overexpressed as in most colon cancers, or it can be lost as in poorly differentiated colon cancers, or it can be "newly" acquired as in squamous cell carcinoma of the esophagus.33Kimura H Kato H Makoto AF Nakajima MS Fukai Y Miyazaki T Masuda N Fukuchi M Kuwano H Prognostic significance of EpCAM expression in human esophageal cancer.Int J Oncol. 2007; 30: 171-179PubMed Google Scholar EpCAM expression has been reported to be a possible marker of early malignancy and has become an important target for immunotherapy with monoclonal antibodies because of its specificity in some cancerous lesions.34Went P Vasei M Bubendorf L Terracciano L Tornillo L Riede U Kononen J Simon R Sauter G Bauerle PA Frequent high-level expression of the immunotherapeutic target Ep-CAM in colon, stomach, prostate and lung cancers.Br J Cancer. 2006; 94: 128-135Crossref PubMed Scopus (309) Google Scholar, 35Baeuerle PA Gires O EpCAM (CD326) finding its role in cancer.Br J Cancer. 2007; 96: 417-423Crossref PubMed Scopus (397) Google Scholar In prostate, EpCAM does not appear to be regulated by androgens; however, the heterogeneous distribution of EpCAM expression within DU145 and PC3 prostatic cell lines suggests that EpCAM expression may be lost in a subpopulation of cells within androgen independent prostate cancer, indicative of the overall loss of cellular differentiation with progression.36Poczatek RB Myers RB Manne U Oelschlager DK Weiss HL Bostwick DG Grizzle WE Ep-CAM levels in prostatic adenocarcinoma and prostatic intraepithelial neoplasia.J Urol. 1999; 162: 1462-1466Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar Studies of EpCAM expression in normal and tumor prostate epithelium have generated conflicting results. One group found up-regulation of EpCAM in hormone-refractory and untreated prostate cancer epithelial cells, but this was not confirmed by another study that examined hormone-refractory cancers, localized cancers, and metastases.31Went P Dirnhofer S Schopf D Moch H Spizzo G Expression and prognostic significance of EpCAM.J Cancer Molecules. 2008; 3: 169-174Google Scholar Moreover, of three analyses of the relationship between EpCAM levels in tumor epithelium and Gleason score, two found no correlation whereas a third study found a positive correlation of EpCAM levels and tumor grade. Finally, a tissue microarray-based study showed up-regulation of EpCAM staining in tumors.37Zellweger T Ninck C Bloch M Mirlacher M Koivisto PA Helin HJ Mihatsch MJ Gasser TC Bubendorf L Expression patterns of potential therapeutic targets in prostate cancer.Int J Cancer. 2005; 113: 619-628Crossref PubMed Scopus (141) Google Scholar The present study is the first to report a difference in EPCAM protein levels in tumor versus normal stroma. However, in contrast to our findings, Poczatek et al36Poczatek RB Myers RB Manne U Oelschlager DK Weiss HL Bostwick DG Grizzle WE Ep-CAM levels in prostatic adenocarcinoma and prostatic intraepithelial neoplasia.J Urol. 1999; 162: 1462-1466Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar reported EpCAM immunostaining did not increase significantly in the stroma of formalin-fixed, paraffin-embedded, archival tissue specimens from patients diagnosed with prostatic carcinoma. Possible explanations include differences in tissue processing and the use of different primary anti-EpCAM antibodies. Moreover, the availability of whole mount sections in our study allowed for a thorough three-dimensional analysis of the tumor microenvironment. In some cases EpCAM staining was localized to only a subregion of the tumor field and would not have been easily identified in small specimens due to sampling effects. The use of stromal markers in the tumor microenvironment has been proposed for both diagnostic and therapeutic purposes.38Micke P Ostman A Tumor-stroma interaction: cancer-associated fibroblasts as novel targets in anti-cancer therapy?.Lung Cancer. 2004; 26: 173-185Google Scholar, 39Petrulio CA Kim-Schulze S Kaufman HL The tumor microenvironment and implications for cancer immunotherapy.Expert Opin Biol Ther. 2006; 6: 671-684Crossref PubMed Scopus (33) Google Scholar Possible targets for clinical intervention include cancer-associated fibroblasts, infiltrating macrophages, tumor endothelial cells, and extracellular matrix. In patients with multiple myeloma, bone marrow stroma is being targeted using a proteasome inhibitor to attenuate the disease, and anti-angiogenic drugs (Avastin [bevacizumab] and thalidomide) are being used to target endothelial cells.40Lipton A Future treatment of bone metastasis.Clin Cancer Res. 2006; 12: 6305-6308Crossref Scopus (65) Google Scholar, 41Anderson K Targeted therapy of multiple myeloma based upon tumor-micro environmental interactions.Exp Hematol. 2007; 35: 155-162Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 42Zipori D The mesenchyme in cancer therapy as a target tumor component, effector cell modality and cytokine expression vehicle.Cancer Metastasis Rev. 2006; 25: 459-467Crossref PubMed Scopus (10) Google Scholar A novel immunological approach to colon cancer therapy targets fibroblast activating protein, that is highly expressed by stroma cells of this tumor type.43Hofheinz RD Al-Batran S-E Hartmann F Hartung G Jager D Renner C Tanswell P Kunz U Amelsberg A Kuthan H Stehle G Stromal antigen targeting by a humanized monoclonal antibody: an early phase II trial of sibrotuzumab in patients with metastatic colorectal cancer.Onkologie. 2003; 26: 44-48Crossref PubMed Scopus (231) Google Scholar Recently, disruption of the epithelial-stroma interaction has been demonstrated to decrease carcinoma cell proliferation when it is accomplished through chemotherapeutic agents.44Schedin P Elias A Multistep tumorigenesis and the microenvironment.Breast Cancer Res. 2004; 6: 93-101Crossref PubMed Scopus (82) Google Scholar Similarly, Kammertoens and colleagues45Kammertoens T Schuler T Blanenstein T Immunotherapy: target the stroma to hit the tumor.Trends Mol Med. 2005; 11: 225-231Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar showed that, in addition to direct tumor killing, T-cell-mediated tumor rejection occurs in part due to disruption of stromal infrastructure. Since the stromal compartment is relatively accessible to vascular-delivered agents, up-regulation of targets in the tumor-associated stroma makes them attractive candidates to consider as clinical tools.46Bouzin C Feron O Targeting tumor stroma and exploiting mature tumor vasculature to improve anti-cancer drug delivery.Drug Resist Updat. 2007; 10: 109-120Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 47Hofmeister V Schrama D Anti-cancer therapies targeting the tumor stroma.Cancer Immunol Immunother. 2008; 57: 1-17Crossref PubMed Scopus (133) Google Scholar, 48Lissbrant IF Stattin P Damber JE Bergh A Vascular density is a predictor of cancer-specific survival in prostatic carcinoma.Prostate. 1997; 15: 38-45Crossref Scopus (130) Google Scholar, 49Weidner N Carroll PR Flax J Blumenfeld W Folkman J Tumor angiogenesis correlates with metastasis in invasive prostate carcinoma.Am J Pathol. 1993; 143: 401-409PubMed Google Scholar, 50Jain RK Normalizing tumor vasculature with anti-angiogenic therapy: a new paradigm for combination therapy.Nat Med. 2001; 7: 987-989Crossref PubMed Scopus (1835) Google Scholar Specifically in the case of EpCAM in the stroma of the prostate tumor micro-environment, IHC analysis of CD31 on whole mounts confirmed a close spatial relationship between stromal EpCAM and neovessels, thus providing the basis for targeting of the tumor microenvironment via systemic delivery of anti-EPCAM directed imaging or therapeutic agents (Figure 5). Moreover, although not statistically significant, EpCAM protein expression showed a trend toward increased stromal levels with Gleason grade as its expression in Gleason 4/5 tumors was higher than in Gleason 3 neoplasms. This observation could have particular importance as molecular markers that increase with tumor grade would be useful clinically. However, a larger set of cases will need to be studied to validate this relationship. In summary, emerging data support the concept that the stroma in the tumor microenvironment functions to significantly affect tumorigenesis in epithelial carcinomas. EpCAM mRNA and protein are both up-regulated in prostate tumor stroma and represent potential targets for new diagnostics or therapeutics.
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