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Expression of α5-integrin, α7-integrin, Ε-cadherin, and N-cadherin in localized prostate cancer
Urologic Oncology: Seminars and Original Investigations ] (2015) ∎∎∎–∎∎∎
Original article
Expression of α5-integrin, α7-integrin, Ε-cadherin, and N-cadherin in localized prostate cancer Alexandros Drivalos, M.D.a,*, Michael Chrisofos, M.D., Ph.D.b, Eleni Efstathiou, M.D., Ph.D.c, Amalia Kapranou, M.D., Ph.D.d, Gerasimos Kollaitis, M.D., Ph.D.a, Georgios Koutlis, B.Scd, Nick Antoniou, M.D., Ph.D.a, Dimitrios Karanastasis, M.D., Ph.D.a, Meletios A. Dimopoulos, M.D., Ph.D.c, Aristotelis Bamias, M.D., Ph.D.c a Department of Urology, Athens General Hospital “Elpis,” Athens, Greece 2nd Department of Urology, School of Medicine, Sismanoglio Hospital, University of Athens, Athens, Greece c Department of Clinical Therapeutics, School of Medicine, Alexandra Hospital, University of Athens, Athens, Greece d Department of Anatomopathology, Athens Navy Hospital, Athens, Greece b
Received 25 April 2015; received in revised form 14 July 2015; accepted 27 October 2015
Abstract Objective: To explore the correlation between the expression of α5-integrin, α7-integrin, Ε-cadherin, and N-cadherin in prostate cancer (PCa) and its clinicopathological data including tumor grade and clinical stage. Methods: The expression of α5-integrin, α7-integrin, Ε-cadherin, and N-cadherin was examined in 157 cases of PCa and adjacent normal prostatic tissue by immunohistochemical assay, and the correlation with clinicopathological features was analyzed. Results: Expressions of α5-integrin, α7-integrin, and Ε-cadherin in PCa were lower than those in normal prostatic tissues (P o 0.05). N-cadherin expression was higher in cancer prostatic tissue than in normal prostatic tissues (P o 0.05). The reduced expression of α5integrin, α7-integrin, and Ε-cadherin was related to Gleason score, pathological stage, lymph node metastasis, and prostate-specific antigen level, but it was not associated with positive surgical margins and patient age. The increased expression of N-cadherin was related to Gleason score, pathological stage, lymph node metastasis, and prostate-specific antigen level, but not to age and positive surgical margins. The expression of E-cadherin was highly negatively correlated with that of N-cadherin and also positively correlated with that of α5-integrin and α7-integrin. Conclusion: The reduced expression of α5-integrin, α7-integrin, and Ε-cadherin and abnormal expression of N-cadherin play an important role in the occurrence and development of PCa. The results indicate that these have potential values in the diagnosis and are predictable indices in the proliferation of PCa. r 2015 Elsevier Inc. All rights reserved.
Keywords: Cadherins; Integrins; Cell adhesion molecules; Prostate cancer
1. Introduction Prostate cancer (PCa) is the most commonly diagnosed male cancer and the second leading cause of cancer-related deaths in Western men [1]. During PCa progression, precursor lesions eventually progress to incurable castration-resistant metastatic disease [2]. Among the alterations described in prostate carcinogenesis are aberrant interactions between glandular epithelial cells and the extracellular matrix, mediated by cell adhesion Corresponding author. Tel.: þ30-693-233-2545. E-mail address: [email protected] (A. Drivalos). *
http://dx.doi.org/10.1016/j.urolonc.2015.10.016 1078-1439/r 2015 Elsevier Inc. All rights reserved.
molecules (CAMs). Several studies have reported dysregulation of various CAMs in localized or metastatic PCa [3]. However, none of these studies had a large number of specimens and was able to define clearly their prognostic value. The most well-characterized and accepted predictors to determine which clinically localized cancers will recur after radical prostatectomy are model equations that take into account preoperative serum prostate-specific antigen (PSA), final Gleason score, and final pathologic stage [4]. Prediction of progression for the individual patient using these statistical models, however, is still not precise, and these models could still be improved on. Thus, additional markers
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are needed to more accurately target high-risk patients for inclusion in clinical trials involving investigational therapies for locally advanced prostate carcinoma. Several other approaches show promise in this regard, including nuclear morphometry, where the results have been quite consistent. Other more controversial markers include DNA ploidy [5] and other biomarkers, such as the amount of tumor angiogenesis [6], and immunohistochemical levels of various markers, including cadherins and integrins [7]. The aim of this study was to assess the expression of a panel of CAMs (α5 integrin, α7 integrin, E-cadherin, and N-cadherin) with reference to known clinicopathological prognostic factors in patients with PCa after radical prostatectomy. 2. Material and methods 2.1. Pathologic specimens Tissue specimens were obtained from 157 patients with PCa who underwent radical prostatectomy at the Athens Navy Hospital between 2002 and 2009. Inclusion criteria were no evidence of metastatic disease in preoperative bone scan and CT scan of the abdomen and pelvis, no preoperative hormonal therapy, chemotherapy or radiotherapy. Serum PSA level before surgical treatment, histopathological differentiation of PCa, capsular penetration, seminal vesicles invasion, involvement of surgical margins, and presence of lymph node metastasis were recorded. The entire prostate was cut into 5mm transverse serial sections. One slice of the whole prostate was fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned at 5 μm. Sections were stained with hematoxylin and eosin for mapping of cancer-affected areas. The histopathological differentiation of PCa was determined according to the Gleason system. Histopathological diagnosis was made by a single pathologist to ensure consistency. 2.2. Tissue microarray Tissue microarrays (TMAs) were constructed from the 157 prostatectomy specimens. Noncancer regions of the samples were used as controls in separate TMA. From the individual radical prostatectomy specimens, were selected areas representing all histologic tumor patterns present. Each case was represented by a median of 60.6 mm-diameter cores (range, 4-9 cores) with 270 cores obtained on each slide. The expression of biomarkers was assessed by immunohistochemical staining in the tumor microenvironment. The stainings were performed with an automated staining instrument (Dako Autostainer-Dako North America, Inc., Carpinteria, CA) using 3,3 diaminobenzidine as the chromogen. 2.3. Immunohistochemical method A total of 4-μm tissue sections cut from formalin-fixed paraffin-embedded TMAs were subjected to
immunohistochemical studies. Immunohistochemical studies were performed using the Dako autostainer (Dako North America Inc., Carpinteria, CA). The sections of tissue were dewaxed with xylene and then rehydrated in progressively decreasing concentrations of alcohol. All samples were then processed according to standard procedures. Primary antibodies (dilution, company) used included the following: E-cadherin (1:100, Dako, Carpinteria, CA), N-cadherin (1:50, Dako, Carpinteria, CA), α5-integrin (1:200, Abcam, USA), and α7-integrin (1:500, Abcam, USA). Antibody binding was detected with a DAB kit (Dako) in which 3,30 diaminobenzidine was the chromogen, and the sections were then counterstained with hematoxylin. Images of each biomarker in each core of the TMA were acquired by using a Bacus Laboratories, Inc. Slide Scanner (BLISS) imaging system and automatically stored for later retrieval (Bacus Laboratories, Inc., Lombard, IL). Immunostaining was evaluated by 2 independent pathologists. A semiquantitative scoring was used for grading the molecules expression based on positive cell percentage and staining intensity. The positive cell percentage was scored 0 to 3 from o5%, 5% to 25%, 26% to 75%, to 475% in order. The positive staining intensity was scored 0 to 3 with 0 representing negative staining, 1 representing weak staining, 2 representing intermediate staining, and 3 representing strong staining. The grading criteria of the molecules expression was determined by the scores given as sum of positive cell percentage and staining intensity scores: o2 points being “,” 2 to 2.9 being “þ,” 3 to 3.9 points being “þþ,” 44 points being “þþþ.” The symbols “” and “þ” were designated as low expression (negative), and “þþ” and “þþþ” as high expression (positive) [8]. 2.4. Statistical analysis Statistical analysis was carried out with the IBM SPSS Statistics 21 software package for Windows. Correlations between expression of each molecule and clinicopathological parameters was evaluated using the χ2 test. Associations among the molecules were analyzed with the Spearman rank correlation. P o 0.05 was considered significant. 3. Results 3.1. Clinicopathological characteristics The clinicopathological characteristics of 157 study participants are summarized in Table 1. The mean age of the patients was 66 years (range: 50–72). In total, 10 tumors (10/157, 6.4%) could be categorized as well differentiated (Gleason score 3–4), 109 (109/157, 69.4%) as moderately differentiated (Gleason score 5–7), and 38 (38/157, 24.2%) as poorly differentiated (Gleason score 8–10). The median preoperative PSA level was 9.2 ng/ml (range: 1.7–26.0). Pelvic lymph node infiltration was found in 14 (8.9%) at
A. Drivalos et al. / Urologic Oncology: Seminars and Original Investigations ] (2015) 1–8 Table 1 Characteristics of 157 study participants Age, median (range)
66 (50–72) PSA, median (range)
Gleason score, n (%) 3–4 10 (6.4) 5–7 109 (69.4) 8–10 38 (24.2) Stage, n(%) pT2 94 (59.9) pT3 63 (40.1) Pelvic lymph nodes, n (%) Negative 143 (91.1) Positive 14 (14)
9.2 ng/ml (1.7–26.0)
PSA, n (%) o10 μg/l 16 (10.2) 10–20 μg/l 104 (66.2) 20–26 μg/l 37 (23.6) Seminal vesicles, n (%) Positive 18 (11.5) Negative 139 (88.5) Surgical margins, n (%) Negative Positive
143 (91.1) 14 (8.9)
time of surgery. Seminal vesicle invasion was shown in 18 cases (11.5%) and surgical margins in 14 cases (8.9%). The pathological stage at time of surgery was pT2 in 94 cases (59.9%) and pT3 in 63 cases (40.1%). 3.2. Integrin α5 and α7 expression Integrin α5 was mainly expressed οn the cellular membrane (Fig. 1). Benign prostate glandular tissue showed more frequently high membrane expression of α5-integrin compared with that of PCa (82.2% [129/157] vs. 42% [66/ 157], P o 0.001; Table 2). Integrin α5 expression was significantly lower in the serum 420 μg/l group than in the serum PSA r 10 μg/l group (P ¼ 0.017). Also, α5integrin expression was correlated with the clinical pathological factors including clinical stage, Gleason score, lymph node metastasis, and seminal vesicles invasion (P 4 0.05; Table 3). There was no statistically significant correlation between α5-integrin expression, patient's age, and cancer-free surgical margins (P 4 0.05; Table 3). Integrin α7 was also expressed on the cellular membrane (Fig. 2). Integrin α7 expression was significantly lower in the PCa tissue than in the adjacent tissue (84.7% vs. 40.8%, P o 0.001). With an increase in Gleason score, α7-integrin expression tended to decrease (P ¼ 0.001). Low membrane
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expression was correlated with advanced pathological stage, lymph node metastasis, and seminal vesicles invasion (P o 0.05). Integrin α7 expression did not correlate with patient's age and positive surgical margins (P 4 0.05). 3.3. E-cadherin and N-cadherin expression E-cadherin was expressed on the cellular membrane (Fig. 3). Benign prostate glandular tissue showed strong membrane expression of E-cadherin compared with PCa (83.4% [131/157] vs. 42% [66/157], P o 0.05; Table 2). E-cadherin expression was significantly lower in the serum PSA groups of 10 to 20 μg/l and 420 μg/l than in the serum PSA r 10 μg/l group (P o 0.05). In addition, E-cadherin expression was correlated with the clinical pathological factors including clinical stage, Gleason score, lymph node metastasis, and seminal vesicles invasion (P 4 0.05; Table 3). No statistically significant correlation was found between E-cadherin expression, patient's age, and cancer-free surgical margins (P 4 0.05; Table 3). N-cadherin was mainly expressed in the cytoplasm and cellular membrane (Fig. 4). N-cadherin expression was significantly higher in the PCa tissue than in the normal tissue (63.7% vs. 15.3%, P o 0.05). There was significant correlation between the Gleason grade and N-cadherin expression (P ¼ 0.001). In addition, there was a correlation between N-cadherin expression pathological stage, lymph node metastasis, and seminal vesicles invasion (P o 0.05). N-cadherin expression did not correlate with patient's age (P 4 0.05) and positive malignant surgical margins (Table 3). 3.4. Association among cell adhesion markers E-cadherin expression was negatively correlated with N-cadherin expression (r ¼ 0.538, P o 0.001). Integrin α5 and α7 expression was positively correlated with Ecadherin expression (r ¼ 0.190, P ¼ 0.017 and r ¼ 0.186, P ¼ 0.019) and negatively correlated with N-cadherin expression (r ¼ 0.243, P ¼ 0.02 and r ¼ 0.236, P ¼ 0.03). Integrin α5 expression is
Fig. 1. α5-Integrin high expression (A) and low expression (B). (Color version of figure is available online.)
A. Drivalos et al. / Urologic Oncology: Seminars and Original Investigations ] (2015) 1–8
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Table 2 Comparisons of E-cadherin, N-cadherin, α5 integrin, and α7 integrin expression in the prostate cancer tissue and normal tissue Prostate cancer n (%)
E-cadherin N-cadherin α5 Integrin α7 Integrin
Normal tissue n (%)
High
Low
High
Low
66 100 66 64
91 57 91 93
131 24 129 133
26 133 28 24
(42) (63.7) (42.0) (40.8)
(58) (84.7) (58.0) (59.2)
positively correlated with α7-integrin expression (r ¼ 0.922, P ¼ 0.000) (Table 4).
4. Discussion The reduction of interaction of glandular epithelial cell with each other and with extracellular matrix results in the loss of cellular polarity and in altered histologic structure, which is a morphologic hallmark of malignant tumors [9]. E-cadherin, N-cadherin, α5-integrin, and α7-integrin are diverse CAMs that contribute in this interaction and promote cell proliferation, migration, and differentiation [9]. Aberrant expression of these molecules is significantly correlated with cancer progression in patients with PCa [10].
(83.4) (36.3) (82.2) (84.7)
(16.6) (84.7) (17.8) (15.3)
χ2
P value
57.558 76.981 53.707 64.860
o0.001 o0.001 o0.001 o0.001
A previous study [11] reported that down-regulation of E-cadherin expression in the epithelial tumor tissue is common and correlates with tumor grading. Strong evidence exists that E-cadherin expression basically disappears or is greatly down-regulated in poorly differentiated tumor cells, such as breast cancer [12], stomach cancer [13], liver cancer [14], and rectal cancer [15]. Several studies have shown that E-cadherin expression is down-regulated in PCa but only in a limited number of participants [16,17]. The results from this study that it has been performed in a larger number of patients have also shown that E-cadherin expression was significantly lower in the PCa tissue than in the normal. Moreover, low E-cadherin expression has been correlated with advanced clinical stage, higher Gleason score, and lymph node metastasis (P o 0.05). The significant correlation of E-cadherin down-regulation with
Table 3 Correlations of E-cadherin, N-cadherin, α5 integrin, and α7 integrin expression levels in the prostate cancer tissue and clinical pathological factors Pathological factors
E-Cadherin
α5 Integrin
N-Cadherin
α7 Integrin
High (%)
Low (%)
P value
High (%)
Low (%)
P value
High (%)
Low (%)
P value
High (%)
Low (%)
P value
Pathological stage pT2 pT3
64.5 30.2
35.5 69.8
0.001
53.2 79.4
46.8 20.6
0.001
64.9 7.9
35.1 92.1
0.000
62.8 7.9
37.2 92.1
0.000
PSA o10 μg/l 10–20 μg/l 20–26 μg/l
68.8 43.3 27.0
31.3 56.7 73.0
0.017
25.0 67.3 70.3
75.0 32.7 29.7
0.003
75.0 43.3 24.3
25.0 56.7 75.7
0.003
68.8 42.3 24.3
31.3 57.7 75.7
0.009
Gleason score 3–4 5–7 8–10
80 44 26.3
20 56 73.7
0.007
10 66.1 71.1
90 33.9 28.9
0.001
80 45 23.7
20 55 76.3
0.003
70 44 23.7
30 56 76.3
0.013
Lymphatic metastasis Positive 14.3 Negative 43.4
85.7 56.6
0.035
85.7 61.5
14.3 38.5
0.013
7.1 45.5
92.9 54.5
0.006
14.3 43.4
85.7 56.6
0.035
Seminal vesicles invasion Positive 16.7 Negative 45.3
83.3 54.7
0.020
88.9 60.4
11.1 39.6
0.018
5.6 94.4
46.8 53.2
0.001
5.6 45.3
94.4 54.7
0.001
Surgical margins Positive Negative
21.4 44.1
78.6 55.9
0.102
85.7 61.5
14.3 38.5
0.073
21.4 45.5
78.6 54.5
0.083
21.4 44.1
78.6 55.9
0.102
Age (years old) r66 466
45.0 39.0
55.0 61.0
0.443
65.0 62.3
35.0 37.7
0.729
42.5 41.6
57.5 58.4
0.905
42.5 39.0
57.5 61.0
0.652
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Fig. 2. α7-Integrin high expression (A) and low expression (B). (Color version of figure is available online.)
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 PCa, which remains to be validated by other studies [18]. At present, the expression and significance of E-cadherin in the PCa is still open to debate. The loss of E-cadherin can be seen as an alteration, in the early stage of PCa, related to the epithelial-mesenchymal transition phenomenon [19]. This phenomenon is a biological program required for the acquisition of malignant traits by carcinoma cells and is related to the loss of the cell adhesion associated with the epithelial phenotype. This change leads to the conversion of the tumor cells to a more migratory, mesenchymallike state and is considered a necessary step for neoplastic dissemination. However, after dissemination and metastasis, tumor cells colonize and grow in a new area, during which, E-cadherin expression is needed, and tumor cells likely reexpress prior down-regulated E-cadherin to promote the growth and expansion of metastatic tumor cells in the newly colonized area.
Ιntegrin α5 binds to matrix macromolecules and proteinases. It is the primary receptor for fibronectin. Several studies report aberrant expression of α5-integrin in tumor cells, such as cervical cancer [20], breast cancer [21], and myeloid leukemia [22]. To our knowledge, few studies report weaker or even absent expression of α5-integrin in PCa compared with normal prostatic tissues. The results in our study, also, demonstrate a lower α5-integrin expression in PCa, which is correlated with higher Gleason score and advanced pathological stage (P o 0.005) in a larger sample. A recent study has demonstrated that α5 integrin turnover is dependent on fibronectin matrix assembly, where the absence of matrix-capable fibronectin in the extracellular space targets the internalized receptor for rapid degradation [23]. These findings could explain the decrease of cell-cell stability, leading to desquamation of an increased proportion of local cells and easily resulting in tumor cell infiltration and metastasis. Ιntegrin α7 functions as a receptor for the basement membrane protein laminin-1. A recent study has demonstrated that mutations are correlated with lower expression of α7-integrin in PCa, liver cancer, glioblastoma multiforme, and leiomyosarcoma [24]. To our knowledge, this is the first report in which the down-regulation of integrin α7
Fig. 3. E-cadherin high expression (A) and low expression (B). (Color version of figure is available online.)
A. Drivalos et al. / Urologic Oncology: Seminars and Original Investigations ] (2015) 1–8
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Fig. 4. N-cadherin high expression (A) and low expression (B). (Color version of figure is available online.)
is correlated with clinicopathological factors. The data from the current study show that α7-integrin expression is strongly down-regulated in PCa compared with benign tissue, and the down-regulation correlates with the increase in Gleason score, higher clinical stage, and lymph mode metastasis. The function of integrin α7 in prostate gland and smooth muscle appears to be related to the adhesion of cells to the basement membrane and prevention of the random migration of these cells to other organs. Another important function of integrin α7 appears to be its role in limiting cell proliferation because expression of integrin α7 induced the expression of proteins that inhibit cell cycle progression and cell growth. When the level of integrin α7 protein was decreased or the protein was mutated, cells appeared to lose inhibitory signals for both cell migration and proliferation.
This loss may lead to unchecked tumor cell proliferation and a higher incidence of metastases. Thus, impairing the function of integrin α7 may be an efficient mechanism of carcinogenesis. N-cadherin is only expressed in neuroectodermal and mesodermic tissues, for example, mature muscle and nerve and hemopoietic tissue, but hardly expressed in normal epithelia cells. There are several studies that report an increase in expression of N-cadherin in PCa cells [25,26]. Similarly, in our study, there is an up-regulation of N-cadherin expression in PCa and is correlated with higher Gleason score and a more advanced clinical stage. Moreover, the results from this study have demonstrated that with the decrease in the degree of differentiation of PCa cells, E-cadherin expression is significantly decreased,
Table 4 Correlation of E-cadherin, N-cadherin, α5 integrin, and α7 integrin in the prostate cancer tissue Cadherin E (n)
Integrin α5 (n)
Cadherin N (n)
Integrin α7 (n)
Low
High
Low
High
Low
High
Low
High
Cadherin E (n) Low High r P
91 0 1.000 0.001
0 66
13 44 0.538 0.001
78 22
60 31 0.190 0.017
31 35
61 32 0.186 0.019
30 34
Cadherin N (n) Low High r P
13 78 0.538 0.001
44 22
57 0 1.000 0.001
0 100
24 67 0.243 0.002
33 33
25 68 0.236 0.003
32 32
Integrin α5 (n) Low High r P
60 31 0.190 0.017
31 35
24 33 0.243 0.002
67 33
91 0 1.000 0.001
0 66
89 4 0.922 0.001
2 62
Integrin α7 (n) Low High r P
61 30 0.186 0.019
32 34
25 32 0.236 0.003
68 32
89 2 0.922 0.001
4 62
93 0 1.000 0.001
0 64
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whereas N-cadherin expression is significantly increased (P o 0.05). The Spearman rank correlation analysis results showed that E-cadherin expression was highly negatively correlated with N-cadherin expression, indicating that the cadherin switches from E-cadherin to N-cadherin in PCa. This “cadherin switch” has been already proposed from Hazan et al. [27]. The precise mechanism has not been fully understood but it is considered that “cadherin switch” is one of important mechanisms underlying epithelial-mesenchymal transition. “Cadherin switch” refers to cadherin transformation from type E to type N during normal cell transformation into malignant cells. Our results also showed that α5-integrin, α7-integrin, E-cadherin, and N-cadherin expression is related to serum PSA level. When serum PSA level increases, E-cadherin, α5-integrin, and α7-integrin expression is decreased, whereas N-cadherin expression is increased. Serum PSA level is an important index of detecting PCa and patients with higher level of serum PSA level exhibit poorer prognosis than patients with lower level of serum PSA [28]. We considered that the abnormal expression of these molecules decrease cell-cell adhesion, destroy cell morphology and integrity, and alter prostate cell permeability, which may contribute to the increase in serum PSA level, further revealing that these changes are related to the occurrence of PCa. Finally, we showed that E-cadherin expression was positively correlated with α5-integrin and α7-integrin expression, suggesting that more CAMs combined might add larger prognostic information than one alone. The aforementioned results are based in a semiquantitative method for the evaluation of the expression of the CAMs, as it is proposed from many pathologists [8,29]. Α more quantitative method, widely accepted and standardized, could consolidate our results.
5. Conclusion Our goal in this study was to correlate α5-integrin, α7integrin, E-cadherin, and N-cadherin expression to known clinicopathological factors as surrogate markers. The limitation of the study is the lack of follow-up data on clinical outcomes, such as PSA recurrence. Despite this apparent limitation, the current study identifies significant alterations in the level of expression, as well as a correlation of these CAMs with known clinicopathological factors such as Gleason score and pathological stage in human PC tissues. These findings suggest that α5-integrin, α7-integrin, Ecadherin, and N-cadherin could be used as novel biomarkers predicting the biological aggressiveness of PCa. References [1] Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J Clin 2012;62:10–29.
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[26] Gravdal K1, Halvorsen OJ, Haukaas SA, Akslen LA. A switch from E-cadherin to N-cadherin expression indicates epithelial to mesenchymal transition and is of strong and independent importance for the progress of prostate cancer. Clin Cancer Res 2007;13: 7003–11. [27] Hazan RB, Qiao R, Keren R, Badano I, Suyama K. Cadherin switch in tumor progression. Ann N Y Acad Sci 2004;1014:155–63. [28] Nadler RB, Loeb S, Roehl KA, Antenor JA, Catalona WJ, Suarez BK, et al. Use of 2.6 ng/mL prostate specific antigen prompt for biopsy in men older than 60 years. J Urol 2005;174:21542157. [29] Jaggi M1, Johansson SL, Baker JJ, Smith LM, Galich A, Balaji KC. Aberrant expression of E-cadherin and beta-catenin in human prostate cancer. Urol Oncol 2005;23:402–6.
Original article
Expression of α5-integrin, α7-integrin, Ε-cadherin, and N-cadherin in localized prostate cancer Alexandros Drivalos, M.D.a,*, Michael Chrisofos, M.D., Ph.D.b, Eleni Efstathiou, M.D., Ph.D.c, Amalia Kapranou, M.D., Ph.D.d, Gerasimos Kollaitis, M.D., Ph.D.a, Georgios Koutlis, B.Scd, Nick Antoniou, M.D., Ph.D.a, Dimitrios Karanastasis, M.D., Ph.D.a, Meletios A. Dimopoulos, M.D., Ph.D.c, Aristotelis Bamias, M.D., Ph.D.c a Department of Urology, Athens General Hospital “Elpis,” Athens, Greece 2nd Department of Urology, School of Medicine, Sismanoglio Hospital, University of Athens, Athens, Greece c Department of Clinical Therapeutics, School of Medicine, Alexandra Hospital, University of Athens, Athens, Greece d Department of Anatomopathology, Athens Navy Hospital, Athens, Greece b
Received 25 April 2015; received in revised form 14 July 2015; accepted 27 October 2015
Abstract Objective: To explore the correlation between the expression of α5-integrin, α7-integrin, Ε-cadherin, and N-cadherin in prostate cancer (PCa) and its clinicopathological data including tumor grade and clinical stage. Methods: The expression of α5-integrin, α7-integrin, Ε-cadherin, and N-cadherin was examined in 157 cases of PCa and adjacent normal prostatic tissue by immunohistochemical assay, and the correlation with clinicopathological features was analyzed. Results: Expressions of α5-integrin, α7-integrin, and Ε-cadherin in PCa were lower than those in normal prostatic tissues (P o 0.05). N-cadherin expression was higher in cancer prostatic tissue than in normal prostatic tissues (P o 0.05). The reduced expression of α5integrin, α7-integrin, and Ε-cadherin was related to Gleason score, pathological stage, lymph node metastasis, and prostate-specific antigen level, but it was not associated with positive surgical margins and patient age. The increased expression of N-cadherin was related to Gleason score, pathological stage, lymph node metastasis, and prostate-specific antigen level, but not to age and positive surgical margins. The expression of E-cadherin was highly negatively correlated with that of N-cadherin and also positively correlated with that of α5-integrin and α7-integrin. Conclusion: The reduced expression of α5-integrin, α7-integrin, and Ε-cadherin and abnormal expression of N-cadherin play an important role in the occurrence and development of PCa. The results indicate that these have potential values in the diagnosis and are predictable indices in the proliferation of PCa. r 2015 Elsevier Inc. All rights reserved.
Keywords: Cadherins; Integrins; Cell adhesion molecules; Prostate cancer
1. Introduction Prostate cancer (PCa) is the most commonly diagnosed male cancer and the second leading cause of cancer-related deaths in Western men [1]. During PCa progression, precursor lesions eventually progress to incurable castration-resistant metastatic disease [2]. Among the alterations described in prostate carcinogenesis are aberrant interactions between glandular epithelial cells and the extracellular matrix, mediated by cell adhesion Corresponding author. Tel.: þ30-693-233-2545. E-mail address: [email protected] (A. Drivalos). *
http://dx.doi.org/10.1016/j.urolonc.2015.10.016 1078-1439/r 2015 Elsevier Inc. All rights reserved.
molecules (CAMs). Several studies have reported dysregulation of various CAMs in localized or metastatic PCa [3]. However, none of these studies had a large number of specimens and was able to define clearly their prognostic value. The most well-characterized and accepted predictors to determine which clinically localized cancers will recur after radical prostatectomy are model equations that take into account preoperative serum prostate-specific antigen (PSA), final Gleason score, and final pathologic stage [4]. Prediction of progression for the individual patient using these statistical models, however, is still not precise, and these models could still be improved on. Thus, additional markers
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are needed to more accurately target high-risk patients for inclusion in clinical trials involving investigational therapies for locally advanced prostate carcinoma. Several other approaches show promise in this regard, including nuclear morphometry, where the results have been quite consistent. Other more controversial markers include DNA ploidy [5] and other biomarkers, such as the amount of tumor angiogenesis [6], and immunohistochemical levels of various markers, including cadherins and integrins [7]. The aim of this study was to assess the expression of a panel of CAMs (α5 integrin, α7 integrin, E-cadherin, and N-cadherin) with reference to known clinicopathological prognostic factors in patients with PCa after radical prostatectomy. 2. Material and methods 2.1. Pathologic specimens Tissue specimens were obtained from 157 patients with PCa who underwent radical prostatectomy at the Athens Navy Hospital between 2002 and 2009. Inclusion criteria were no evidence of metastatic disease in preoperative bone scan and CT scan of the abdomen and pelvis, no preoperative hormonal therapy, chemotherapy or radiotherapy. Serum PSA level before surgical treatment, histopathological differentiation of PCa, capsular penetration, seminal vesicles invasion, involvement of surgical margins, and presence of lymph node metastasis were recorded. The entire prostate was cut into 5mm transverse serial sections. One slice of the whole prostate was fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned at 5 μm. Sections were stained with hematoxylin and eosin for mapping of cancer-affected areas. The histopathological differentiation of PCa was determined according to the Gleason system. Histopathological diagnosis was made by a single pathologist to ensure consistency. 2.2. Tissue microarray Tissue microarrays (TMAs) were constructed from the 157 prostatectomy specimens. Noncancer regions of the samples were used as controls in separate TMA. From the individual radical prostatectomy specimens, were selected areas representing all histologic tumor patterns present. Each case was represented by a median of 60.6 mm-diameter cores (range, 4-9 cores) with 270 cores obtained on each slide. The expression of biomarkers was assessed by immunohistochemical staining in the tumor microenvironment. The stainings were performed with an automated staining instrument (Dako Autostainer-Dako North America, Inc., Carpinteria, CA) using 3,3 diaminobenzidine as the chromogen. 2.3. Immunohistochemical method A total of 4-μm tissue sections cut from formalin-fixed paraffin-embedded TMAs were subjected to
immunohistochemical studies. Immunohistochemical studies were performed using the Dako autostainer (Dako North America Inc., Carpinteria, CA). The sections of tissue were dewaxed with xylene and then rehydrated in progressively decreasing concentrations of alcohol. All samples were then processed according to standard procedures. Primary antibodies (dilution, company) used included the following: E-cadherin (1:100, Dako, Carpinteria, CA), N-cadherin (1:50, Dako, Carpinteria, CA), α5-integrin (1:200, Abcam, USA), and α7-integrin (1:500, Abcam, USA). Antibody binding was detected with a DAB kit (Dako) in which 3,30 diaminobenzidine was the chromogen, and the sections were then counterstained with hematoxylin. Images of each biomarker in each core of the TMA were acquired by using a Bacus Laboratories, Inc. Slide Scanner (BLISS) imaging system and automatically stored for later retrieval (Bacus Laboratories, Inc., Lombard, IL). Immunostaining was evaluated by 2 independent pathologists. A semiquantitative scoring was used for grading the molecules expression based on positive cell percentage and staining intensity. The positive cell percentage was scored 0 to 3 from o5%, 5% to 25%, 26% to 75%, to 475% in order. The positive staining intensity was scored 0 to 3 with 0 representing negative staining, 1 representing weak staining, 2 representing intermediate staining, and 3 representing strong staining. The grading criteria of the molecules expression was determined by the scores given as sum of positive cell percentage and staining intensity scores: o2 points being “,” 2 to 2.9 being “þ,” 3 to 3.9 points being “þþ,” 44 points being “þþþ.” The symbols “” and “þ” were designated as low expression (negative), and “þþ” and “þþþ” as high expression (positive) [8]. 2.4. Statistical analysis Statistical analysis was carried out with the IBM SPSS Statistics 21 software package for Windows. Correlations between expression of each molecule and clinicopathological parameters was evaluated using the χ2 test. Associations among the molecules were analyzed with the Spearman rank correlation. P o 0.05 was considered significant. 3. Results 3.1. Clinicopathological characteristics The clinicopathological characteristics of 157 study participants are summarized in Table 1. The mean age of the patients was 66 years (range: 50–72). In total, 10 tumors (10/157, 6.4%) could be categorized as well differentiated (Gleason score 3–4), 109 (109/157, 69.4%) as moderately differentiated (Gleason score 5–7), and 38 (38/157, 24.2%) as poorly differentiated (Gleason score 8–10). The median preoperative PSA level was 9.2 ng/ml (range: 1.7–26.0). Pelvic lymph node infiltration was found in 14 (8.9%) at
A. Drivalos et al. / Urologic Oncology: Seminars and Original Investigations ] (2015) 1–8 Table 1 Characteristics of 157 study participants Age, median (range)
66 (50–72) PSA, median (range)
Gleason score, n (%) 3–4 10 (6.4) 5–7 109 (69.4) 8–10 38 (24.2) Stage, n(%) pT2 94 (59.9) pT3 63 (40.1) Pelvic lymph nodes, n (%) Negative 143 (91.1) Positive 14 (14)
9.2 ng/ml (1.7–26.0)
PSA, n (%) o10 μg/l 16 (10.2) 10–20 μg/l 104 (66.2) 20–26 μg/l 37 (23.6) Seminal vesicles, n (%) Positive 18 (11.5) Negative 139 (88.5) Surgical margins, n (%) Negative Positive
143 (91.1) 14 (8.9)
time of surgery. Seminal vesicle invasion was shown in 18 cases (11.5%) and surgical margins in 14 cases (8.9%). The pathological stage at time of surgery was pT2 in 94 cases (59.9%) and pT3 in 63 cases (40.1%). 3.2. Integrin α5 and α7 expression Integrin α5 was mainly expressed οn the cellular membrane (Fig. 1). Benign prostate glandular tissue showed more frequently high membrane expression of α5-integrin compared with that of PCa (82.2% [129/157] vs. 42% [66/ 157], P o 0.001; Table 2). Integrin α5 expression was significantly lower in the serum 420 μg/l group than in the serum PSA r 10 μg/l group (P ¼ 0.017). Also, α5integrin expression was correlated with the clinical pathological factors including clinical stage, Gleason score, lymph node metastasis, and seminal vesicles invasion (P 4 0.05; Table 3). There was no statistically significant correlation between α5-integrin expression, patient's age, and cancer-free surgical margins (P 4 0.05; Table 3). Integrin α7 was also expressed on the cellular membrane (Fig. 2). Integrin α7 expression was significantly lower in the PCa tissue than in the adjacent tissue (84.7% vs. 40.8%, P o 0.001). With an increase in Gleason score, α7-integrin expression tended to decrease (P ¼ 0.001). Low membrane
3
expression was correlated with advanced pathological stage, lymph node metastasis, and seminal vesicles invasion (P o 0.05). Integrin α7 expression did not correlate with patient's age and positive surgical margins (P 4 0.05). 3.3. E-cadherin and N-cadherin expression E-cadherin was expressed on the cellular membrane (Fig. 3). Benign prostate glandular tissue showed strong membrane expression of E-cadherin compared with PCa (83.4% [131/157] vs. 42% [66/157], P o 0.05; Table 2). E-cadherin expression was significantly lower in the serum PSA groups of 10 to 20 μg/l and 420 μg/l than in the serum PSA r 10 μg/l group (P o 0.05). In addition, E-cadherin expression was correlated with the clinical pathological factors including clinical stage, Gleason score, lymph node metastasis, and seminal vesicles invasion (P 4 0.05; Table 3). No statistically significant correlation was found between E-cadherin expression, patient's age, and cancer-free surgical margins (P 4 0.05; Table 3). N-cadherin was mainly expressed in the cytoplasm and cellular membrane (Fig. 4). N-cadherin expression was significantly higher in the PCa tissue than in the normal tissue (63.7% vs. 15.3%, P o 0.05). There was significant correlation between the Gleason grade and N-cadherin expression (P ¼ 0.001). In addition, there was a correlation between N-cadherin expression pathological stage, lymph node metastasis, and seminal vesicles invasion (P o 0.05). N-cadherin expression did not correlate with patient's age (P 4 0.05) and positive malignant surgical margins (Table 3). 3.4. Association among cell adhesion markers E-cadherin expression was negatively correlated with N-cadherin expression (r ¼ 0.538, P o 0.001). Integrin α5 and α7 expression was positively correlated with Ecadherin expression (r ¼ 0.190, P ¼ 0.017 and r ¼ 0.186, P ¼ 0.019) and negatively correlated with N-cadherin expression (r ¼ 0.243, P ¼ 0.02 and r ¼ 0.236, P ¼ 0.03). Integrin α5 expression is
Fig. 1. α5-Integrin high expression (A) and low expression (B). (Color version of figure is available online.)
A. Drivalos et al. / Urologic Oncology: Seminars and Original Investigations ] (2015) 1–8
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Table 2 Comparisons of E-cadherin, N-cadherin, α5 integrin, and α7 integrin expression in the prostate cancer tissue and normal tissue Prostate cancer n (%)
E-cadherin N-cadherin α5 Integrin α7 Integrin
Normal tissue n (%)
High
Low
High
Low
66 100 66 64
91 57 91 93
131 24 129 133
26 133 28 24
(42) (63.7) (42.0) (40.8)
(58) (84.7) (58.0) (59.2)
positively correlated with α7-integrin expression (r ¼ 0.922, P ¼ 0.000) (Table 4).
4. Discussion The reduction of interaction of glandular epithelial cell with each other and with extracellular matrix results in the loss of cellular polarity and in altered histologic structure, which is a morphologic hallmark of malignant tumors [9]. E-cadherin, N-cadherin, α5-integrin, and α7-integrin are diverse CAMs that contribute in this interaction and promote cell proliferation, migration, and differentiation [9]. Aberrant expression of these molecules is significantly correlated with cancer progression in patients with PCa [10].
(83.4) (36.3) (82.2) (84.7)
(16.6) (84.7) (17.8) (15.3)
χ2
P value
57.558 76.981 53.707 64.860
o0.001 o0.001 o0.001 o0.001
A previous study [11] reported that down-regulation of E-cadherin expression in the epithelial tumor tissue is common and correlates with tumor grading. Strong evidence exists that E-cadherin expression basically disappears or is greatly down-regulated in poorly differentiated tumor cells, such as breast cancer [12], stomach cancer [13], liver cancer [14], and rectal cancer [15]. Several studies have shown that E-cadherin expression is down-regulated in PCa but only in a limited number of participants [16,17]. The results from this study that it has been performed in a larger number of patients have also shown that E-cadherin expression was significantly lower in the PCa tissue than in the normal. Moreover, low E-cadherin expression has been correlated with advanced clinical stage, higher Gleason score, and lymph node metastasis (P o 0.05). The significant correlation of E-cadherin down-regulation with
Table 3 Correlations of E-cadherin, N-cadherin, α5 integrin, and α7 integrin expression levels in the prostate cancer tissue and clinical pathological factors Pathological factors
E-Cadherin
α5 Integrin
N-Cadherin
α7 Integrin
High (%)
Low (%)
P value
High (%)
Low (%)
P value
High (%)
Low (%)
P value
High (%)
Low (%)
P value
Pathological stage pT2 pT3
64.5 30.2
35.5 69.8
0.001
53.2 79.4
46.8 20.6
0.001
64.9 7.9
35.1 92.1
0.000
62.8 7.9
37.2 92.1
0.000
PSA o10 μg/l 10–20 μg/l 20–26 μg/l
68.8 43.3 27.0
31.3 56.7 73.0
0.017
25.0 67.3 70.3
75.0 32.7 29.7
0.003
75.0 43.3 24.3
25.0 56.7 75.7
0.003
68.8 42.3 24.3
31.3 57.7 75.7
0.009
Gleason score 3–4 5–7 8–10
80 44 26.3
20 56 73.7
0.007
10 66.1 71.1
90 33.9 28.9
0.001
80 45 23.7
20 55 76.3
0.003
70 44 23.7
30 56 76.3
0.013
Lymphatic metastasis Positive 14.3 Negative 43.4
85.7 56.6
0.035
85.7 61.5
14.3 38.5
0.013
7.1 45.5
92.9 54.5
0.006
14.3 43.4
85.7 56.6
0.035
Seminal vesicles invasion Positive 16.7 Negative 45.3
83.3 54.7
0.020
88.9 60.4
11.1 39.6
0.018
5.6 94.4
46.8 53.2
0.001
5.6 45.3
94.4 54.7
0.001
Surgical margins Positive Negative
21.4 44.1
78.6 55.9
0.102
85.7 61.5
14.3 38.5
0.073
21.4 45.5
78.6 54.5
0.083
21.4 44.1
78.6 55.9
0.102
Age (years old) r66 466
45.0 39.0
55.0 61.0
0.443
65.0 62.3
35.0 37.7
0.729
42.5 41.6
57.5 58.4
0.905
42.5 39.0
57.5 61.0
0.652
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Fig. 2. α7-Integrin high expression (A) and low expression (B). (Color version of figure is available online.)
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 PCa, which remains to be validated by other studies [18]. At present, the expression and significance of E-cadherin in the PCa is still open to debate. The loss of E-cadherin can be seen as an alteration, in the early stage of PCa, related to the epithelial-mesenchymal transition phenomenon [19]. This phenomenon is a biological program required for the acquisition of malignant traits by carcinoma cells and is related to the loss of the cell adhesion associated with the epithelial phenotype. This change leads to the conversion of the tumor cells to a more migratory, mesenchymallike state and is considered a necessary step for neoplastic dissemination. However, after dissemination and metastasis, tumor cells colonize and grow in a new area, during which, E-cadherin expression is needed, and tumor cells likely reexpress prior down-regulated E-cadherin to promote the growth and expansion of metastatic tumor cells in the newly colonized area.
Ιntegrin α5 binds to matrix macromolecules and proteinases. It is the primary receptor for fibronectin. Several studies report aberrant expression of α5-integrin in tumor cells, such as cervical cancer [20], breast cancer [21], and myeloid leukemia [22]. To our knowledge, few studies report weaker or even absent expression of α5-integrin in PCa compared with normal prostatic tissues. The results in our study, also, demonstrate a lower α5-integrin expression in PCa, which is correlated with higher Gleason score and advanced pathological stage (P o 0.005) in a larger sample. A recent study has demonstrated that α5 integrin turnover is dependent on fibronectin matrix assembly, where the absence of matrix-capable fibronectin in the extracellular space targets the internalized receptor for rapid degradation [23]. These findings could explain the decrease of cell-cell stability, leading to desquamation of an increased proportion of local cells and easily resulting in tumor cell infiltration and metastasis. Ιntegrin α7 functions as a receptor for the basement membrane protein laminin-1. A recent study has demonstrated that mutations are correlated with lower expression of α7-integrin in PCa, liver cancer, glioblastoma multiforme, and leiomyosarcoma [24]. To our knowledge, this is the first report in which the down-regulation of integrin α7
Fig. 3. E-cadherin high expression (A) and low expression (B). (Color version of figure is available online.)
A. Drivalos et al. / Urologic Oncology: Seminars and Original Investigations ] (2015) 1–8
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Fig. 4. N-cadherin high expression (A) and low expression (B). (Color version of figure is available online.)
is correlated with clinicopathological factors. The data from the current study show that α7-integrin expression is strongly down-regulated in PCa compared with benign tissue, and the down-regulation correlates with the increase in Gleason score, higher clinical stage, and lymph mode metastasis. The function of integrin α7 in prostate gland and smooth muscle appears to be related to the adhesion of cells to the basement membrane and prevention of the random migration of these cells to other organs. Another important function of integrin α7 appears to be its role in limiting cell proliferation because expression of integrin α7 induced the expression of proteins that inhibit cell cycle progression and cell growth. When the level of integrin α7 protein was decreased or the protein was mutated, cells appeared to lose inhibitory signals for both cell migration and proliferation.
This loss may lead to unchecked tumor cell proliferation and a higher incidence of metastases. Thus, impairing the function of integrin α7 may be an efficient mechanism of carcinogenesis. N-cadherin is only expressed in neuroectodermal and mesodermic tissues, for example, mature muscle and nerve and hemopoietic tissue, but hardly expressed in normal epithelia cells. There are several studies that report an increase in expression of N-cadherin in PCa cells [25,26]. Similarly, in our study, there is an up-regulation of N-cadherin expression in PCa and is correlated with higher Gleason score and a more advanced clinical stage. Moreover, the results from this study have demonstrated that with the decrease in the degree of differentiation of PCa cells, E-cadherin expression is significantly decreased,
Table 4 Correlation of E-cadherin, N-cadherin, α5 integrin, and α7 integrin in the prostate cancer tissue Cadherin E (n)
Integrin α5 (n)
Cadherin N (n)
Integrin α7 (n)
Low
High
Low
High
Low
High
Low
High
Cadherin E (n) Low High r P
91 0 1.000 0.001
0 66
13 44 0.538 0.001
78 22
60 31 0.190 0.017
31 35
61 32 0.186 0.019
30 34
Cadherin N (n) Low High r P
13 78 0.538 0.001
44 22
57 0 1.000 0.001
0 100
24 67 0.243 0.002
33 33
25 68 0.236 0.003
32 32
Integrin α5 (n) Low High r P
60 31 0.190 0.017
31 35
24 33 0.243 0.002
67 33
91 0 1.000 0.001
0 66
89 4 0.922 0.001
2 62
Integrin α7 (n) Low High r P
61 30 0.186 0.019
32 34
25 32 0.236 0.003
68 32
89 2 0.922 0.001
4 62
93 0 1.000 0.001
0 64
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whereas N-cadherin expression is significantly increased (P o 0.05). The Spearman rank correlation analysis results showed that E-cadherin expression was highly negatively correlated with N-cadherin expression, indicating that the cadherin switches from E-cadherin to N-cadherin in PCa. This “cadherin switch” has been already proposed from Hazan et al. [27]. The precise mechanism has not been fully understood but it is considered that “cadherin switch” is one of important mechanisms underlying epithelial-mesenchymal transition. “Cadherin switch” refers to cadherin transformation from type E to type N during normal cell transformation into malignant cells. Our results also showed that α5-integrin, α7-integrin, E-cadherin, and N-cadherin expression is related to serum PSA level. When serum PSA level increases, E-cadherin, α5-integrin, and α7-integrin expression is decreased, whereas N-cadherin expression is increased. Serum PSA level is an important index of detecting PCa and patients with higher level of serum PSA level exhibit poorer prognosis than patients with lower level of serum PSA [28]. We considered that the abnormal expression of these molecules decrease cell-cell adhesion, destroy cell morphology and integrity, and alter prostate cell permeability, which may contribute to the increase in serum PSA level, further revealing that these changes are related to the occurrence of PCa. Finally, we showed that E-cadherin expression was positively correlated with α5-integrin and α7-integrin expression, suggesting that more CAMs combined might add larger prognostic information than one alone. The aforementioned results are based in a semiquantitative method for the evaluation of the expression of the CAMs, as it is proposed from many pathologists [8,29]. Α more quantitative method, widely accepted and standardized, could consolidate our results.
5. Conclusion Our goal in this study was to correlate α5-integrin, α7integrin, E-cadherin, and N-cadherin expression to known clinicopathological factors as surrogate markers. The limitation of the study is the lack of follow-up data on clinical outcomes, such as PSA recurrence. Despite this apparent limitation, the current study identifies significant alterations in the level of expression, as well as a correlation of these CAMs with known clinicopathological factors such as Gleason score and pathological stage in human PC tissues. These findings suggest that α5-integrin, α7-integrin, Ecadherin, and N-cadherin could be used as novel biomarkers predicting the biological aggressiveness of PCa. References [1] Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J Clin 2012;62:10–29.
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