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Aberrant expression of E-cadherin and beta-catenin in human prostate cancer☆
Urologic Oncology: Seminars and Original Investigations 23 (2005) 402– 406
Original article
Aberrant expression of E-cadherin and beta-catenin in human prostate cancer夡 Meena Jaggi, Ph.D.*, Sonny L. Johansson, M.D., Ph.D., John J. Baker, M.D., Lynette M. Smith, M.S., Anton Galich, M.D., K.C. Balaji, M.D., F.R.C.S.* University of Nebraska Medical Center, Omaha, NE 68198-2360, USA Received 24 December 2004; received in revised form 22 March 2005; accepted 23 March 2005
Abstract Cadherin-catenin complexes play a key role in embryonic development, and are associated with carcinogenesis and metastasis. We studied the expression of the major members of the family, including E-cadherin and -catenin in prostate cancer (PC), and correlated with Gleason grade and pathologic stage. Immunohistochemistry was performed on serial sections of paraffinized radical prostatectomy specimens to evaluate E-cadherin (n ⫽ 16) and -catenin (n ⫽ 17) expression using heat induced epitope retrieval. Benign appearing prostate epithelium was used as an internal control in each specimen. Two pathologists independently reviewed and scored the intensity and extent of immunostaining using a semiquantitative scale. The Mantel-Haenszel method, stratified by reviewer, was used to test for an association among Gleason score, pathologic stage, and the expression of E-cadherin or -catenin in PC. Gleason grade ⱖ7 cancers showed significantly lower expression of E-cadherin and -catenin compared to Gleason grade ⬍7 PC, P ⫽ 0.015 and 0.025, respectively. In addition, -catenin was down regulated in 4 of 5 (80%) specimens with identifiable high-grade prostatic intraepithelial neoplasia and had demonstrable nuclear staining in higher grade PC (P ⫽ 0.0001). However, E-cadherin and -catenin membranous or nuclear expressions were not significantly associated with final pathologic stage of the specimens (P values ⬎0.05). Overall, the expression of E-cadherin and -catenin is significantly down regulated in PC compared to surrounding benign appearing prostate, which correlates with increasing Gleason grade. Furthermore, nuclear localization of -catenin in high grade PC may be a useful biomarker for aggressive PC. © 2005 Elsevier Inc. All rights reserved. Keywords: Beta-catenin; E-cadherin; Cell adhesion molecules; Prostate cancer; Gleason grade
1. Introduction Prostate cancer (PC) is the most commonly diagnosed noncutaneous cancer and is the second leading cause of cancer-related deaths in American men [1,2]. Virtually all patients dying of PC have metastasis at the time of death [3]. Cancer cell metastasis is influenced by the cell’s ability to migrate from the primary site and thrive in the microenvironment of a secondary site. The cadherin family of transmembrane glycoprotein plays a critical role in cell-to-cell adhesion, and cadherin dysregulation is strongly associated with cancer metastasis and progression [4]. The inter-epi-
夡 This work was supported by a seed grant from the University of Nebraska Foundation, Omaha, NE. * Corresponding authors. Tel.: ⫹1-402-559-4292; fax: ⫹1-402-5596529. E-mail address: [email protected] (M. Jaggi).
1078-1439/05/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.urolonc.2005.03.024
thelial binding of E-cadherin mediates lateral cell-cell adhesion in secretory tissues such as the prostate and mammary gland, and results in the formation of adherens junctions that are required for tissue morphogenesis and maintenance of the differentiated phenotype [5]. Cadherin binds to ␣, , and ␥-catenin [6], which physically associate with actin filaments and the actin cytoskeleton [7]. Loss of immunoreactivity of the normal membranous E-cadherin and catenin complex is common in transitional cell carcinoma of bladder and correlates with high-grade, advanced stage, and poor prognosis [8]. Because the cadherin/catenin system plays a major and complex role in cancer progression, we studied the expression of the major members of the family, including E-cadherin and -catenin in PC. Several studies have reported dysregulation of cadherin or catenin protein expression in localized or metastatic PC. Although aberrant E-cadherin expression significantly correlates with prostate-specific antigen (PSA) recurrence in
M. Jaggi et al. / Urologic Oncology: Seminars and Original Investigations 23 (2005) 402– 406
patients undergoing radical prostatectomy for PC [9,10], correlation with Gleason grade has not been clearly shown [11–13]. In this study, we compared the expression of Ecadherin and -catenin between benign and malignant prostate tissues within the same specimen. Because the Gleason grading system is most frequently used to grade PC and is strongly associated with stage of the disease and clinical progression, we explored the correlation of E-cadherin and -catenin expression with Gleason grade. In addition, we directly correlated the E-cadherin and -catenin expression with final pathologic stage of the prostatectomy specimens.
403
analyzed using an Eclipse E-400 microscope (Nikon Corp., Tokyo, Japan), and representative photographs were taken. Two pathologists (S.L.J. and J.J.B.) independently scored the E-cadherin and -catenin tissue staining. An ordinate, categorical (semiquantitative) method was used to assess staining across the whole of each section (tumor and benign area). Cadherin expression was graded as: 0 ⫽ no staining; 1⫹ ⫽ weak positive staining; 2⫹ ⫽ moderate staining; and 3⫹ ⫽ strong membrane bound staining, uniformly visualized over the whole section. 2.4. Statistical analysis
2. Materials and methods 2.1. Human PC tissue Formalin fixed paraffin-embedded, archival radical prostatectomy specimens were obtained from our institutional pathology department after institutional review board approval. 2.2. Antibodies and reagents Unless otherwise stated, all reagents were obtained from Sigma Chemical Company (St. Louis, MO). 2.3. Immunohistochemistry of human PC tissue Serial sections of paraffinized radical prostatectomy specimens were processed for immunohistochemical analysis of E-cadherin and -catenin expression in PC using heat induced epitope retrieval. One of the serial sections was stained with hematoxylin and eosin to distinguish benign from cancerous tissue, and to determine the Gleason grade [14]. Paraffin embedded tissue sections (4 m) were deparaffinized in xylene, rehydrated, washed in phosphate buffered saline (PBS), treated with 0.3% H2O2 in absolute methanol for 30 minutes to quench endogenous peroxide activity, placed in hot citrate buffer (pH 6.0), and heated in a microwave oven at 700 W for 15⫺30 minutes. After antigen retrieval, the tissue sections were stained using the Vector ABC kit (Vector Laboratories, Burlingame, CA) as described by the manufacturer. Briefly, the slides were washed with PBS, treated with horse serum for 30 minutes at room temperature to block nonspecific binding, incubated with antimouse monoclonal E-cadherin (HECD1) or -catenin (6F9) antibodies for one hour at room temperature, washed with PBS-T (PBS with 0.05% Tween-20), incubated with a secondary antibody for 30 minutes at room temperature, washed again (3 ⫻ 5 minutes) with PBS-T before incubation with the ABC solution. The reaction color was developed by treating the tissue sections with 3, 3-diaminobenzidine (Vector Laboratories). The slides were washed with water, counterstained with hematoxylin, dehydrated, and mounted with Vectamount permanent mounting media (Vector Laboratories). All slides were
SAS software (SAS Institute Inc., Cary, NC) was used for statistical analysis. We limited the sample size of PC specimens to 16 and 17 for E-cadherin and -catenin, respectively, because statistically significant dysregulation of these proteins was shown at the given sample size, and further increase in sample numbers will not have altered the final conclusions. To measure the strength of agreement between the 2 reviewers, kappa measures were used for the staining of benign and malignant glands. Kappa equals zero (0) when the agreement equals that expected by chance, and it equals 1 when there is perfect agreement (⬍0.4, poor; 0.4 – 0.75, good; and ⬎0.75, excellent agreement). The Mantel-Haenszel method, stratified by reviewer, was used to test for an association between Gleason score and pathologic stage with the expression of E-cadherin or -catenin in PC. The P values have not been adjusted for multiple comparisons, and values ⬍0.05 are considered statistically significant.
3. Results 3.1. E-cadherin expression A total of 16 PC specimens were analyzed, comprising 8 samples (50%) of Gleason grade 2⫺6 and 8 samples (50%) of Gleason grade 7⫺10. Benign prostate glandular tissue showed strong membrane staining (3⫹) of E-cadherin compared to PC within the same tissue section. PC sections showed a consistent decrease in expression of E-cadherin in glandular epithelia (Fig. 1A). Overall, we obtained good agreement between the 2 reviewers on E-cadherin expression in malignant tissues (kappa 0.43, 95% confidence interval 0.07⫺0.78). However, in patients with Gleason grade 2⫺6 PC, reviewer 2 scored absence of E-cadherin staining in 1 specimen compared to 4 specimens by reviewer 1 because the weak staining seen was considered background by the latter. E-cadherin expression was significantly down regulated in Gleason grade 7⫺10 PC compared to Gleason grade ⱕ6 PC (P ⫽ 0.015) (Table 1), suggesting a significant association of E-cadherin expression with increasing grade. Although our study shows a correlation between E-cadherin down-regulation with increasing grade, the findings need to
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M. Jaggi et al. / Urologic Oncology: Seminars and Original Investigations 23 (2005) 402– 406
Fig. 1. E-cadherin and -catenin expression in human PC specimens. (A) Decreased E-cadherin staining in malignant glands (arrowhead) compared to benign appearing glands (arrow). (B) The -catenin immunostaining in human PC specimen shows decreased membranous expression of -catenin in malignant glands (arrowhead) compared to benign appearing glands (arrow). (C) High-grade PIN showing decreased -catenin expression as compared to benign appearing glands (Fig. 1B arrow). (D) High-grade Gleason prostate carcinoma; 9 (4 ⫹ 5) showing nuclear staining of -catenin.
be validated using a larger cohort. We also correlated the E-cadherin expression with the pathologic stage of these samples. Of 16 samples, 14 (87.5%) were organ confined (stage ⱕT2), and 2 (12.5%) were nonorgan confined (stage ⱖT3). There was no significant correlation between pathologic stage and E-cadherin expression in this study (P ⫽ 0.31) (Table 2).
3.2. -catenin expression A total of 17 samples of PC specimens were analyzed comprising 9 (53%) with Gleason grade 2⫺6 and 8 samples (47%) with Gleason grade 7⫺10 tumors. There was complete agreement for strong -catenin staining (3⫹) of benign prostatic tissue and good agreement (kappa ⫽ 0.58, 95% confidence interval 0.17, 0.98) for staining of PC tissue between both reviewers. Most of the PC specimens showed
Table 1 Association of E-cadherin expression with Gleason grade Reviewer
1
2
E-cadherin score 0 1⫹ ⬎1⫹ 0 1⫹ ⬎1⫹
Number of Gleason grade 2–6 (%)
Number of Gleason grade 7–10 (%)
4 (50) 1 (13) 3 (37) 1 (13) 6 (75) 1 (13)
0 (0) 7 (87) 1 (13) 0 (0) 7 (87) 1 (13)
P Value
0.015
Table 2 Association of E-cadherin expression with pathologic stage Reviewer
E-cadherin score
Number of stage T1–T2 (%)
Number of stage T3–T40 (%)
1
0–1⫹ ⬎1⫹ 0–1⫹ ⬎1⫹
10 (71) 4 (29) 12 (86) 2 (14)
2 (100) 0 2 (100) 0
2
P Value
0.31
M. Jaggi et al. / Urologic Oncology: Seminars and Original Investigations 23 (2005) 402– 406 Table 3 Association of membranous -catenin expression with Gleason grade Reviewer
-Catenin score
Number of Gleason grade 2–6 (%)
Number of Gleason grade 7–10 (%)
1
1⫹ ⬎1⫹ 1⫹ ⬎1⫹
1 (11) 8 (89) 0 9 (100)
3 (37) 5 (63) 3 (37) 5 (63)
2
P Value
Reduction in cell-cell adhesion results in the loss of cellular polarity and in altered histologic structure, which is the morphologic hallmark of malignant tumors [15]. Ecadherin is a tumor suppressor gene product concentrated in the adherens junctions on lateral cell borders linking the cytoskeleton of neighboring cells through the catenin complex of proteins [15]. Aberrant E-cadherin expression is significantly correlated with PSA recurrence in patients undergoing radical prostatectomy for PC [9]. Table 4 Association of nuclear -catenin expression with Gleason grade
-Catenin score
Number of Gleason grade 2–6 (%)
Number of Gleason grade 7–10 (%)
1
0 ⱖ1⫹ 0 ⱖ1⫹
9 (100) 0 9 (100) 0
3 (37) 5 (63) 3 (37) 5 (63)
2
Reviewer
-Catenin score
Number of stage T1–T2 (%)
Number of stage T1–T2 (%)
1
1⫹ ⬎1⫹ 1⫹ ⬎1⫹
5 (31) 11 (69) 3 (19) 13 (81)
0 1 (100) 0 1 (100)
2
4. Discussion
Reviewer
Table 5 Association of membranous -catenin expression with pathologic stage
0.025
significant down-regulation of membranous -catenin expression in PC compared to benign prostatic glands (Fig. 1B). There was also a significant association of reduced membranous -catenin expression with increasing Gleason grade (P ⫽ 0.025) (Table 3). Although our study shows a correlation between membranous -catenin down-regulation and increasing grade, the finding needs to be validated using a larger cohort. Interestingly, 4 of 5 samples with high-grade prostatic intraepithelial neoplasia (PIN) showed significant down-regulation of -catenin compared to benign prostatic glands (Fig. 1C), suggesting that down-regulation of -catenin may be an early event in prostatic carcinogenesis. Moreover, -catenin was localized in the nucleus of poorly differentiated cancer cells of Gleason grade 7⫺10 (Fig. 1D). There was also significant correlation between higher Gleason grade and nuclear -catenin expression in PC (P ⫽ 0.0001) (Table 4). Of 17 PC specimens, 16 (94%) were organ confined (stage ⱕT2), and 1 (6%) was nonorgan confined (stage T4). However, there was no significant correlation of membranous (P ⫽ 0.31) (Table 5) or nuclear -catenin expression (P ⫽ 0.43) (Table 6) with the pathologic stage of PC specimens.
405
P Value
0.43
The data from the current study show that E-cadherin expression is strongly down regulated in PC compared to benign appearing prostate in the same tissue section, and the down-regulation correlates with increasing Gleason grade. The significant correlation of E-cadherin down-regulation with increasing Gleason grade in this study provides additional evidence for the role for E-cadherin in PC progression. Although down-regulation of E-cadherin is validated in progressive PC and is well established as an early event in prostate carcinogenesis, one study in the literature has shown strong E-cadherin expression in metastatic PC, which remains to be validated by other studies [16]. Significant down-regulation of E-cadherin and -catenin in localized PC and putative activating mutations (missense and deletion) at a rate of 5% has been previously shown [17,18]. In addition to showing down-regulation of -catenin and E-cadherin, our study also identified that -catenin is down regulated independent of E-cadherin in 4 of 5 (80%) of specimens with identifiable PIN. Although the down-regulation of -catenin in preneoplastic lesions of other cancers such as colon and oral tumors is well known, down-regulation of -catenin in PIN is a novel finding in PC [19,20]. Down-regulation of -catenin in PIN remains to be validated using a larger sample set. Another hallmark of -catenin activity in cells is nuclear localization. Translocation of -catenin to the nucleus is associated with increased transcription of target genes associated with cellular proliferation or cell death [21]. Our study shows a nuclear localization of -catenin in Gleason score ⱖ7 cancers compared to the absence of nuclear -catenin expression in Gleason score ⱕ6 PC (P ⫽ 0.0001). Although the functional significance of this finding remains to be studied, it is conceivable that altered -catenin expression and location contribute to PC progression because
Table 6 Association of nuclear -catenin expression with pathologic stage P Value
Reviewer
-Catenin score
Number of stage T1–T2 (%)
Number of stage T1–T2 (%)
1
0 ⱖ1⫹ 0 ⱖ1⫹
11 (69) 5 (31) 11 (69) 5 (31)
1 (100) 0 1 (100) 0
0.0001 2
P Value
0.36
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-catenin is linked to androgen signaling, cell proliferation, and cell death. The down-regulation of E-cadherin in PC may result in reduced -catenin binding to E-cadherin, releasing the unbound -catenin to activate other pathways, as evidenced by significantly higher nuclear translocation of -catenin in high grade PC. Yang et al. [22] have elegantly showed that expression of E-cadherin in E-cadherin-negative PC cells results in redistribution of the cytoplasmic -catenin to the cell membrane and a reduction in androgen receptor mediated transcription, which suggests a potential mechanistic and functional basis for altered -catenin expression in PC. The Gleason grading system is a well established grading system in PC that correlates with pathologic stage, disease progression, and recurrence [14]. Although both E-cadherin and -catenin expressions correlated with Gleason grade in PC, our study failed to show significant correlation with pathologic stage of the specimens. This result may related to the small sample size of the study containing very few nonorgan confined PC specimens. Alternatively, alteration in E-cadherin and -catenin expression may be markers of early carcinogenesis, which may not directly influence the pathologic stage of the disease at radical prostatectomy. Because the study was performed using radical prostatectomy specimens instead of the prostate biopsy specimens and patients undergoing radical prostatectomy are more likely to have organ confined disease, the lack of correlation between E-cadherin and -catenin expression with pathologic stage may be a result of selection bias. Our goal in this study was to correlate cadherin-catenin protein complex expression to Gleason grade as a surrogate marker. The limitation of the study is the lack of follow-up data on clinical outcomes such as tumor stage or PSA recurrence. Despite these apparent limitations, the current study identifies significant alterations in the level of expression, as well as subcellular localization of E-cadherin and -catenin in human PC tissues stratified by Gleason grade.
5. Conclusions Our study shows that both E-cadherin and -catenin are down regulated in human PC, and significantly correlate with increasing Gleason grade. The -catenin is also down regulated in 4 of 5 (80%) of PIN cases, and nuclear translocation occurs in high-grade PC, suggesting that alterations in -catenin location in PC cells may contribute to cancer progression. Overall, the alteration in the cadherin-catenin complex of proteins suggests a functionally significant role in prostatic carcinogenesis and progression. In particular, specific alternations in -catenin expression in PIN suggest that -catenin can be an early biomarker in PC.
Acknowledgments The authors thank Dr. Masotoshi Takechi, Kyoto University, Kyoto, Japan, and Drs. Margaret J. Wheelock and Keith R. Johnson, University of Nebraska Medical Center, Omaha, NE, for generously providing the mouse monoclonal antibody against human E-cadherin (HECD1) and -catenin (6F9). References [1] Hellerstedt BA, Pienta KJ. The current state of hormonal therapy for prostate cancer. CA Cancer J Clin 2002;52:154 –79. [2] Jemal A, Murray T, Samuels A, et al. Cancer statistics, 2003. CA Cancer J Clin 2003;53:5–26. [3] Glass TR, Tangen CM, Crawford ED, et al. Metastatic carcinoma of the prostate: Identifying prognostic groups using recursive partitioning. J Urol 2003;169:164 –9. [4] Yap AS. The morphogenetic role of cadherin cell adhesion molecules in human cancer: A thematic review. Cancer Invest 1998;16:252– 61. [5] Takeichi M. Cadherin cell adhesion receptors as a morphogenetic regulator. Science 1991;251:1451–5. [6] Kemler R. From cadherins to catenins: Cytoplasmic protein interactions and regulation of cell adhesion. Trends Genet 1993;9:317–21. [7] Knudsen KA, Soler AP, Johnson KR, et al. Interaction of alphaactinin with the cadherin/catenin cell-cell adhesion complex via alpha-catenin. J Cell Biol 1995;130:67–77. [8] Okegawa T, Li Y, Pong RC, et al. Cell adhesion proteins as tumor suppressors. J Urol 2002;167:1836 – 43. [9] Rhodes DR, Sanda MG, Otte AP, et al. Multiplex biomarker approach for determining risk of prostate-specific antigen-defined recurrence of prostate cancer. J Natl Cancer Inst 2003;95:661– 8. [10] Wu TT, Hsu YS, Wang JS, et al. The role of p53, bcl-2 and Ecadherin expression in predicting biochemical relapse for organ confined prostate cancer in Taiwan. J Urol 2003;170:78 – 81. [11] Koksal IT, Ozcan F, Kilicaslan I, et al. Expression of E-cadherin in prostate cancer in formalin-fixed, paraffin-embedded tissues: Correlation with pathological features. Pathology 2002;34:233– 8. [12] Busch C, Hanssen TA, Wagener C, et al. Down-regulation of CEACAM1 in human prostate cancer: Correlation with loss of cell polarity, increased proliferation rate, and Gleason grade 3 to 4 transition. Hum Pathol 2002;33:290 – 8. [13] Rubin MA, Mucci NR, Figurski J, et al. E-cadherin expression in prostate cancer: A broad survey using high-density tissue microarray technology. Hum Pathol 2001;32:690 –7. [14] Gleason DF. Histologic grading of prostate cancer: A perspective. Hum Pathol 1992;23:273–9. [15] Hirohashi S, Kanai Y. Cell adhesion system and human cancer morphogenesis. Cancer Sci 2003;94:575– 81. [16] De Marzo AM, Knudsen B, Chan-Tack K, et al. E-cadherin expression as a marker of tumor aggressiveness in routinely processed radical prostatectomy specimens. Urology 1999;53:707–13. [17] Kallakury BV, Sheehan CE, Ross JS. Co-downregulation of cell adhesion proteins alpha- and beta-catenins, p120CTN, E-cadherin, and CD44 in prostatic adenocarcinomas. Hum Pathol 2001;32:849 –55. [18] Chesire DR, Ewing CM, Gage WR, et al. In vitro evidence for complex modes of nuclear beta-catenin signaling during prostate growth and tumorigenesis. Oncogene 2002;21:2679 –94. [19] Valentini AM, Pirrelli M, Renna L, et al. P53 and Beta-catenin in colorectal cancer progression. Curr Pharm Des 2003;9:1932– 6. [20] Sato K, Okazaki Y, Tonogi M, et al. Expression of beta-catenin in rat oral epithelial dysplasia induced by 4-nitroquinoline 1-oxide. Oral Oncol 2002;38:772– 8. [21] Morin PJ. Beta-catenin signaling and cancer. Bioessays 1999;21:1021–30. [22] Yang F, Li X, Sharma M, et al. Linking beta-catenin to androgensignaling pathway. J Biol Chem 2002;277:11336 – 44.
Original article
Aberrant expression of E-cadherin and beta-catenin in human prostate cancer夡 Meena Jaggi, Ph.D.*, Sonny L. Johansson, M.D., Ph.D., John J. Baker, M.D., Lynette M. Smith, M.S., Anton Galich, M.D., K.C. Balaji, M.D., F.R.C.S.* University of Nebraska Medical Center, Omaha, NE 68198-2360, USA Received 24 December 2004; received in revised form 22 March 2005; accepted 23 March 2005
Abstract Cadherin-catenin complexes play a key role in embryonic development, and are associated with carcinogenesis and metastasis. We studied the expression of the major members of the family, including E-cadherin and -catenin in prostate cancer (PC), and correlated with Gleason grade and pathologic stage. Immunohistochemistry was performed on serial sections of paraffinized radical prostatectomy specimens to evaluate E-cadherin (n ⫽ 16) and -catenin (n ⫽ 17) expression using heat induced epitope retrieval. Benign appearing prostate epithelium was used as an internal control in each specimen. Two pathologists independently reviewed and scored the intensity and extent of immunostaining using a semiquantitative scale. The Mantel-Haenszel method, stratified by reviewer, was used to test for an association among Gleason score, pathologic stage, and the expression of E-cadherin or -catenin in PC. Gleason grade ⱖ7 cancers showed significantly lower expression of E-cadherin and -catenin compared to Gleason grade ⬍7 PC, P ⫽ 0.015 and 0.025, respectively. In addition, -catenin was down regulated in 4 of 5 (80%) specimens with identifiable high-grade prostatic intraepithelial neoplasia and had demonstrable nuclear staining in higher grade PC (P ⫽ 0.0001). However, E-cadherin and -catenin membranous or nuclear expressions were not significantly associated with final pathologic stage of the specimens (P values ⬎0.05). Overall, the expression of E-cadherin and -catenin is significantly down regulated in PC compared to surrounding benign appearing prostate, which correlates with increasing Gleason grade. Furthermore, nuclear localization of -catenin in high grade PC may be a useful biomarker for aggressive PC. © 2005 Elsevier Inc. All rights reserved. Keywords: Beta-catenin; E-cadherin; Cell adhesion molecules; Prostate cancer; Gleason grade
1. Introduction Prostate cancer (PC) is the most commonly diagnosed noncutaneous cancer and is the second leading cause of cancer-related deaths in American men [1,2]. Virtually all patients dying of PC have metastasis at the time of death [3]. Cancer cell metastasis is influenced by the cell’s ability to migrate from the primary site and thrive in the microenvironment of a secondary site. The cadherin family of transmembrane glycoprotein plays a critical role in cell-to-cell adhesion, and cadherin dysregulation is strongly associated with cancer metastasis and progression [4]. The inter-epi-
夡 This work was supported by a seed grant from the University of Nebraska Foundation, Omaha, NE. * Corresponding authors. Tel.: ⫹1-402-559-4292; fax: ⫹1-402-5596529. E-mail address: [email protected] (M. Jaggi).
1078-1439/05/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.urolonc.2005.03.024
thelial binding of E-cadherin mediates lateral cell-cell adhesion in secretory tissues such as the prostate and mammary gland, and results in the formation of adherens junctions that are required for tissue morphogenesis and maintenance of the differentiated phenotype [5]. Cadherin binds to ␣, , and ␥-catenin [6], which physically associate with actin filaments and the actin cytoskeleton [7]. Loss of immunoreactivity of the normal membranous E-cadherin and catenin complex is common in transitional cell carcinoma of bladder and correlates with high-grade, advanced stage, and poor prognosis [8]. Because the cadherin/catenin system plays a major and complex role in cancer progression, we studied the expression of the major members of the family, including E-cadherin and -catenin in PC. Several studies have reported dysregulation of cadherin or catenin protein expression in localized or metastatic PC. Although aberrant E-cadherin expression significantly correlates with prostate-specific antigen (PSA) recurrence in
M. Jaggi et al. / Urologic Oncology: Seminars and Original Investigations 23 (2005) 402– 406
patients undergoing radical prostatectomy for PC [9,10], correlation with Gleason grade has not been clearly shown [11–13]. In this study, we compared the expression of Ecadherin and -catenin between benign and malignant prostate tissues within the same specimen. Because the Gleason grading system is most frequently used to grade PC and is strongly associated with stage of the disease and clinical progression, we explored the correlation of E-cadherin and -catenin expression with Gleason grade. In addition, we directly correlated the E-cadherin and -catenin expression with final pathologic stage of the prostatectomy specimens.
403
analyzed using an Eclipse E-400 microscope (Nikon Corp., Tokyo, Japan), and representative photographs were taken. Two pathologists (S.L.J. and J.J.B.) independently scored the E-cadherin and -catenin tissue staining. An ordinate, categorical (semiquantitative) method was used to assess staining across the whole of each section (tumor and benign area). Cadherin expression was graded as: 0 ⫽ no staining; 1⫹ ⫽ weak positive staining; 2⫹ ⫽ moderate staining; and 3⫹ ⫽ strong membrane bound staining, uniformly visualized over the whole section. 2.4. Statistical analysis
2. Materials and methods 2.1. Human PC tissue Formalin fixed paraffin-embedded, archival radical prostatectomy specimens were obtained from our institutional pathology department after institutional review board approval. 2.2. Antibodies and reagents Unless otherwise stated, all reagents were obtained from Sigma Chemical Company (St. Louis, MO). 2.3. Immunohistochemistry of human PC tissue Serial sections of paraffinized radical prostatectomy specimens were processed for immunohistochemical analysis of E-cadherin and -catenin expression in PC using heat induced epitope retrieval. One of the serial sections was stained with hematoxylin and eosin to distinguish benign from cancerous tissue, and to determine the Gleason grade [14]. Paraffin embedded tissue sections (4 m) were deparaffinized in xylene, rehydrated, washed in phosphate buffered saline (PBS), treated with 0.3% H2O2 in absolute methanol for 30 minutes to quench endogenous peroxide activity, placed in hot citrate buffer (pH 6.0), and heated in a microwave oven at 700 W for 15⫺30 minutes. After antigen retrieval, the tissue sections were stained using the Vector ABC kit (Vector Laboratories, Burlingame, CA) as described by the manufacturer. Briefly, the slides were washed with PBS, treated with horse serum for 30 minutes at room temperature to block nonspecific binding, incubated with antimouse monoclonal E-cadherin (HECD1) or -catenin (6F9) antibodies for one hour at room temperature, washed with PBS-T (PBS with 0.05% Tween-20), incubated with a secondary antibody for 30 minutes at room temperature, washed again (3 ⫻ 5 minutes) with PBS-T before incubation with the ABC solution. The reaction color was developed by treating the tissue sections with 3, 3-diaminobenzidine (Vector Laboratories). The slides were washed with water, counterstained with hematoxylin, dehydrated, and mounted with Vectamount permanent mounting media (Vector Laboratories). All slides were
SAS software (SAS Institute Inc., Cary, NC) was used for statistical analysis. We limited the sample size of PC specimens to 16 and 17 for E-cadherin and -catenin, respectively, because statistically significant dysregulation of these proteins was shown at the given sample size, and further increase in sample numbers will not have altered the final conclusions. To measure the strength of agreement between the 2 reviewers, kappa measures were used for the staining of benign and malignant glands. Kappa equals zero (0) when the agreement equals that expected by chance, and it equals 1 when there is perfect agreement (⬍0.4, poor; 0.4 – 0.75, good; and ⬎0.75, excellent agreement). The Mantel-Haenszel method, stratified by reviewer, was used to test for an association between Gleason score and pathologic stage with the expression of E-cadherin or -catenin in PC. The P values have not been adjusted for multiple comparisons, and values ⬍0.05 are considered statistically significant.
3. Results 3.1. E-cadherin expression A total of 16 PC specimens were analyzed, comprising 8 samples (50%) of Gleason grade 2⫺6 and 8 samples (50%) of Gleason grade 7⫺10. Benign prostate glandular tissue showed strong membrane staining (3⫹) of E-cadherin compared to PC within the same tissue section. PC sections showed a consistent decrease in expression of E-cadherin in glandular epithelia (Fig. 1A). Overall, we obtained good agreement between the 2 reviewers on E-cadherin expression in malignant tissues (kappa 0.43, 95% confidence interval 0.07⫺0.78). However, in patients with Gleason grade 2⫺6 PC, reviewer 2 scored absence of E-cadherin staining in 1 specimen compared to 4 specimens by reviewer 1 because the weak staining seen was considered background by the latter. E-cadherin expression was significantly down regulated in Gleason grade 7⫺10 PC compared to Gleason grade ⱕ6 PC (P ⫽ 0.015) (Table 1), suggesting a significant association of E-cadherin expression with increasing grade. Although our study shows a correlation between E-cadherin down-regulation with increasing grade, the findings need to
404
M. Jaggi et al. / Urologic Oncology: Seminars and Original Investigations 23 (2005) 402– 406
Fig. 1. E-cadherin and -catenin expression in human PC specimens. (A) Decreased E-cadherin staining in malignant glands (arrowhead) compared to benign appearing glands (arrow). (B) The -catenin immunostaining in human PC specimen shows decreased membranous expression of -catenin in malignant glands (arrowhead) compared to benign appearing glands (arrow). (C) High-grade PIN showing decreased -catenin expression as compared to benign appearing glands (Fig. 1B arrow). (D) High-grade Gleason prostate carcinoma; 9 (4 ⫹ 5) showing nuclear staining of -catenin.
be validated using a larger cohort. We also correlated the E-cadherin expression with the pathologic stage of these samples. Of 16 samples, 14 (87.5%) were organ confined (stage ⱕT2), and 2 (12.5%) were nonorgan confined (stage ⱖT3). There was no significant correlation between pathologic stage and E-cadherin expression in this study (P ⫽ 0.31) (Table 2).
3.2. -catenin expression A total of 17 samples of PC specimens were analyzed comprising 9 (53%) with Gleason grade 2⫺6 and 8 samples (47%) with Gleason grade 7⫺10 tumors. There was complete agreement for strong -catenin staining (3⫹) of benign prostatic tissue and good agreement (kappa ⫽ 0.58, 95% confidence interval 0.17, 0.98) for staining of PC tissue between both reviewers. Most of the PC specimens showed
Table 1 Association of E-cadherin expression with Gleason grade Reviewer
1
2
E-cadherin score 0 1⫹ ⬎1⫹ 0 1⫹ ⬎1⫹
Number of Gleason grade 2–6 (%)
Number of Gleason grade 7–10 (%)
4 (50) 1 (13) 3 (37) 1 (13) 6 (75) 1 (13)
0 (0) 7 (87) 1 (13) 0 (0) 7 (87) 1 (13)
P Value
0.015
Table 2 Association of E-cadherin expression with pathologic stage Reviewer
E-cadherin score
Number of stage T1–T2 (%)
Number of stage T3–T40 (%)
1
0–1⫹ ⬎1⫹ 0–1⫹ ⬎1⫹
10 (71) 4 (29) 12 (86) 2 (14)
2 (100) 0 2 (100) 0
2
P Value
0.31
M. Jaggi et al. / Urologic Oncology: Seminars and Original Investigations 23 (2005) 402– 406 Table 3 Association of membranous -catenin expression with Gleason grade Reviewer
-Catenin score
Number of Gleason grade 2–6 (%)
Number of Gleason grade 7–10 (%)
1
1⫹ ⬎1⫹ 1⫹ ⬎1⫹
1 (11) 8 (89) 0 9 (100)
3 (37) 5 (63) 3 (37) 5 (63)
2
P Value
Reduction in cell-cell adhesion results in the loss of cellular polarity and in altered histologic structure, which is the morphologic hallmark of malignant tumors [15]. Ecadherin is a tumor suppressor gene product concentrated in the adherens junctions on lateral cell borders linking the cytoskeleton of neighboring cells through the catenin complex of proteins [15]. Aberrant E-cadherin expression is significantly correlated with PSA recurrence in patients undergoing radical prostatectomy for PC [9]. Table 4 Association of nuclear -catenin expression with Gleason grade
-Catenin score
Number of Gleason grade 2–6 (%)
Number of Gleason grade 7–10 (%)
1
0 ⱖ1⫹ 0 ⱖ1⫹
9 (100) 0 9 (100) 0
3 (37) 5 (63) 3 (37) 5 (63)
2
Reviewer
-Catenin score
Number of stage T1–T2 (%)
Number of stage T1–T2 (%)
1
1⫹ ⬎1⫹ 1⫹ ⬎1⫹
5 (31) 11 (69) 3 (19) 13 (81)
0 1 (100) 0 1 (100)
2
4. Discussion
Reviewer
Table 5 Association of membranous -catenin expression with pathologic stage
0.025
significant down-regulation of membranous -catenin expression in PC compared to benign prostatic glands (Fig. 1B). There was also a significant association of reduced membranous -catenin expression with increasing Gleason grade (P ⫽ 0.025) (Table 3). Although our study shows a correlation between membranous -catenin down-regulation and increasing grade, the finding needs to be validated using a larger cohort. Interestingly, 4 of 5 samples with high-grade prostatic intraepithelial neoplasia (PIN) showed significant down-regulation of -catenin compared to benign prostatic glands (Fig. 1C), suggesting that down-regulation of -catenin may be an early event in prostatic carcinogenesis. Moreover, -catenin was localized in the nucleus of poorly differentiated cancer cells of Gleason grade 7⫺10 (Fig. 1D). There was also significant correlation between higher Gleason grade and nuclear -catenin expression in PC (P ⫽ 0.0001) (Table 4). Of 17 PC specimens, 16 (94%) were organ confined (stage ⱕT2), and 1 (6%) was nonorgan confined (stage T4). However, there was no significant correlation of membranous (P ⫽ 0.31) (Table 5) or nuclear -catenin expression (P ⫽ 0.43) (Table 6) with the pathologic stage of PC specimens.
405
P Value
0.43
The data from the current study show that E-cadherin expression is strongly down regulated in PC compared to benign appearing prostate in the same tissue section, and the down-regulation correlates with increasing Gleason grade. The significant correlation of E-cadherin down-regulation with increasing Gleason grade in this study provides additional evidence for the role for E-cadherin in PC progression. Although down-regulation of E-cadherin is validated in progressive PC and is well established as an early event in prostate carcinogenesis, one study in the literature has shown strong E-cadherin expression in metastatic PC, which remains to be validated by other studies [16]. Significant down-regulation of E-cadherin and -catenin in localized PC and putative activating mutations (missense and deletion) at a rate of 5% has been previously shown [17,18]. In addition to showing down-regulation of -catenin and E-cadherin, our study also identified that -catenin is down regulated independent of E-cadherin in 4 of 5 (80%) of specimens with identifiable PIN. Although the down-regulation of -catenin in preneoplastic lesions of other cancers such as colon and oral tumors is well known, down-regulation of -catenin in PIN is a novel finding in PC [19,20]. Down-regulation of -catenin in PIN remains to be validated using a larger sample set. Another hallmark of -catenin activity in cells is nuclear localization. Translocation of -catenin to the nucleus is associated with increased transcription of target genes associated with cellular proliferation or cell death [21]. Our study shows a nuclear localization of -catenin in Gleason score ⱖ7 cancers compared to the absence of nuclear -catenin expression in Gleason score ⱕ6 PC (P ⫽ 0.0001). Although the functional significance of this finding remains to be studied, it is conceivable that altered -catenin expression and location contribute to PC progression because
Table 6 Association of nuclear -catenin expression with pathologic stage P Value
Reviewer
-Catenin score
Number of stage T1–T2 (%)
Number of stage T1–T2 (%)
1
0 ⱖ1⫹ 0 ⱖ1⫹
11 (69) 5 (31) 11 (69) 5 (31)
1 (100) 0 1 (100) 0
0.0001 2
P Value
0.36
406
M. Jaggi et al. / Urologic Oncology: Seminars and Original Investigations 23 (2005) 402– 406
-catenin is linked to androgen signaling, cell proliferation, and cell death. The down-regulation of E-cadherin in PC may result in reduced -catenin binding to E-cadherin, releasing the unbound -catenin to activate other pathways, as evidenced by significantly higher nuclear translocation of -catenin in high grade PC. Yang et al. [22] have elegantly showed that expression of E-cadherin in E-cadherin-negative PC cells results in redistribution of the cytoplasmic -catenin to the cell membrane and a reduction in androgen receptor mediated transcription, which suggests a potential mechanistic and functional basis for altered -catenin expression in PC. The Gleason grading system is a well established grading system in PC that correlates with pathologic stage, disease progression, and recurrence [14]. Although both E-cadherin and -catenin expressions correlated with Gleason grade in PC, our study failed to show significant correlation with pathologic stage of the specimens. This result may related to the small sample size of the study containing very few nonorgan confined PC specimens. Alternatively, alteration in E-cadherin and -catenin expression may be markers of early carcinogenesis, which may not directly influence the pathologic stage of the disease at radical prostatectomy. Because the study was performed using radical prostatectomy specimens instead of the prostate biopsy specimens and patients undergoing radical prostatectomy are more likely to have organ confined disease, the lack of correlation between E-cadherin and -catenin expression with pathologic stage may be a result of selection bias. Our goal in this study was to correlate cadherin-catenin protein complex expression to Gleason grade as a surrogate marker. The limitation of the study is the lack of follow-up data on clinical outcomes such as tumor stage or PSA recurrence. Despite these apparent limitations, the current study identifies significant alterations in the level of expression, as well as subcellular localization of E-cadherin and -catenin in human PC tissues stratified by Gleason grade.
5. Conclusions Our study shows that both E-cadherin and -catenin are down regulated in human PC, and significantly correlate with increasing Gleason grade. The -catenin is also down regulated in 4 of 5 (80%) of PIN cases, and nuclear translocation occurs in high-grade PC, suggesting that alterations in -catenin location in PC cells may contribute to cancer progression. Overall, the alteration in the cadherin-catenin complex of proteins suggests a functionally significant role in prostatic carcinogenesis and progression. In particular, specific alternations in -catenin expression in PIN suggest that -catenin can be an early biomarker in PC.
Acknowledgments The authors thank Dr. Masotoshi Takechi, Kyoto University, Kyoto, Japan, and Drs. Margaret J. Wheelock and Keith R. Johnson, University of Nebraska Medical Center, Omaha, NE, for generously providing the mouse monoclonal antibody against human E-cadherin (HECD1) and -catenin (6F9). References [1] Hellerstedt BA, Pienta KJ. The current state of hormonal therapy for prostate cancer. CA Cancer J Clin 2002;52:154 –79. [2] Jemal A, Murray T, Samuels A, et al. Cancer statistics, 2003. CA Cancer J Clin 2003;53:5–26. [3] Glass TR, Tangen CM, Crawford ED, et al. Metastatic carcinoma of the prostate: Identifying prognostic groups using recursive partitioning. J Urol 2003;169:164 –9. [4] Yap AS. The morphogenetic role of cadherin cell adhesion molecules in human cancer: A thematic review. Cancer Invest 1998;16:252– 61. [5] Takeichi M. Cadherin cell adhesion receptors as a morphogenetic regulator. Science 1991;251:1451–5. [6] Kemler R. From cadherins to catenins: Cytoplasmic protein interactions and regulation of cell adhesion. Trends Genet 1993;9:317–21. [7] Knudsen KA, Soler AP, Johnson KR, et al. Interaction of alphaactinin with the cadherin/catenin cell-cell adhesion complex via alpha-catenin. J Cell Biol 1995;130:67–77. [8] Okegawa T, Li Y, Pong RC, et al. Cell adhesion proteins as tumor suppressors. J Urol 2002;167:1836 – 43. [9] Rhodes DR, Sanda MG, Otte AP, et al. Multiplex biomarker approach for determining risk of prostate-specific antigen-defined recurrence of prostate cancer. J Natl Cancer Inst 2003;95:661– 8. [10] Wu TT, Hsu YS, Wang JS, et al. The role of p53, bcl-2 and Ecadherin expression in predicting biochemical relapse for organ confined prostate cancer in Taiwan. J Urol 2003;170:78 – 81. [11] Koksal IT, Ozcan F, Kilicaslan I, et al. Expression of E-cadherin in prostate cancer in formalin-fixed, paraffin-embedded tissues: Correlation with pathological features. Pathology 2002;34:233– 8. [12] Busch C, Hanssen TA, Wagener C, et al. Down-regulation of CEACAM1 in human prostate cancer: Correlation with loss of cell polarity, increased proliferation rate, and Gleason grade 3 to 4 transition. Hum Pathol 2002;33:290 – 8. [13] Rubin MA, Mucci NR, Figurski J, et al. E-cadherin expression in prostate cancer: A broad survey using high-density tissue microarray technology. Hum Pathol 2001;32:690 –7. [14] Gleason DF. Histologic grading of prostate cancer: A perspective. Hum Pathol 1992;23:273–9. [15] Hirohashi S, Kanai Y. Cell adhesion system and human cancer morphogenesis. Cancer Sci 2003;94:575– 81. [16] De Marzo AM, Knudsen B, Chan-Tack K, et al. E-cadherin expression as a marker of tumor aggressiveness in routinely processed radical prostatectomy specimens. Urology 1999;53:707–13. [17] Kallakury BV, Sheehan CE, Ross JS. Co-downregulation of cell adhesion proteins alpha- and beta-catenins, p120CTN, E-cadherin, and CD44 in prostatic adenocarcinomas. Hum Pathol 2001;32:849 –55. [18] Chesire DR, Ewing CM, Gage WR, et al. In vitro evidence for complex modes of nuclear beta-catenin signaling during prostate growth and tumorigenesis. Oncogene 2002;21:2679 –94. [19] Valentini AM, Pirrelli M, Renna L, et al. P53 and Beta-catenin in colorectal cancer progression. Curr Pharm Des 2003;9:1932– 6. [20] Sato K, Okazaki Y, Tonogi M, et al. Expression of beta-catenin in rat oral epithelial dysplasia induced by 4-nitroquinoline 1-oxide. Oral Oncol 2002;38:772– 8. [21] Morin PJ. Beta-catenin signaling and cancer. Bioessays 1999;21:1021–30. [22] Yang F, Li X, Sharma M, et al. Linking beta-catenin to androgensignaling pathway. J Biol Chem 2002;277:11336 – 44.