Cadherin Switching in Human Prostate Cancer Progression
Kyoichi Tomita, Adrie van Bokhoven, Geert J. L. H. van Leenders, Emiel T. G. Ruijter, Cornelius F. J. Jansen,Marion J. G. Bussemakers, and Jack A. Schalken1
Urology Research Laboratory [K. T., A. v. B., C. F. J. J., M. J. G. B., J. A. S.] and Department of Pathology [G. J. L. H. v. L., E. T. G. R.], University Hospital Nijmegen, 6500 HBNijmegen, the Netherlands
ABSTRACT
The progression of carcinomas is associated with the loss of epithelialmorphology and a concomitant acquisition of a more mesenchymal phe-notype, which in turn is thought to contribute to the invasive and/ormetastatic behavior of the malignant process. Changes in the expression ofcadherins, “cadherin switching,” plays a critical role during embryogen-esis, particularly in morphogenetic processes. Loss of E-cadherin is re-ported to be associated with a poor prognosis; however, thus far, evidence(R. Umbas,et al., Cancer Res.54: 3929–3933, 1994) for up-regulation ofother cadherins has only been reportedin vitro, i.e., we have foundevidence (M. J. G. Bussemakerset al., Int. J. Cancer,85: 446–450, 2000)for cadherin switching in prostate cancer cell lines (up-regulation ofN-cadherin and cadherin-11, two mesenchymal cadherins, in cell lines thatlack a functional E-cadherin-catenin adhesion complex). Here, we reporton the immunohistochemical analysis of the expression of N-cadherin andcadherin-11 in human prostate cancer specimens. N-cadherin was notexpressed in normal prostate tissue; however, in prostatic cancer,N-cadherin was found to be expressed in the poorly differentiated areas,which showed mainly aberrant or negative E-cadherin staining. Cad-herin-11 is expressed in the stroma of all prostatic tumors, in the areawhere stromal and epithelial cells are found. In addition, cadherin-11 isalso expressed in a dotted pattern or at the membrane of the epithelialcells of high-grade cancers. In a number of metastatic lesions, N-cadherinand cadherin-11 are expressed homogeneously. These data raise the pos-sibility that cadherin switching plays an important role in prostate cancermetastasis.
INTRODUCTION
For patients with prostate cancer, it is now well documented thatdecreased expression of E-cadherin is associated with a poor progno-sis (1, 2). The biological functional relation between loss of E-cadherin expression and acquisition of invasive behavior has beendescribed by several groups (3–6), and, moreover, the restoration ofan epithelial phenotype and a concomitant reduction in invasivenessafter DNA-mediated transfection of E-cadherin has been reported byseveral independent investigators (6, 7). The clinical data that show inseveral epithelial-derived malignancies that the loss of E-cadherinexpression is not only associated with invasiveness but also withmetastatic behavior and poor clinical outcome (1, 2, 8–11) suggestthat this event is pivotal in the complex metastatic cascade (12).Clearly, the mere loss of cell-cell contact and communication cannotbe the sole explanation for the observed relation between loss ofE-cadherin-mediated adhesion and poor clinical outcome.
Recently, a number of papers have been published that describe theinappropriate expression of nonepithelial cadherins by epithelial cellsas a putative novel mechanism for promoting the interaction with thestroma, thereby facilitating invasion and metastasis (13–16). Indeed,we were able to show that human prostate cancer cell lines, which lackexpression of either E-cadherin or the catenins and, therefore, lack an
E-cadherin-mediated cell-cell adhesion, show expression of severalother cadherins (17). Most prominent is the expression of the mesen-chymal N-cadherin and cadherin-11 in these cell lines. Moreover, inother epithelial-tissue derived cancer cell lines,e.g.,human bladder,melanoma, and breast cancer cell lines (15, 16, 18–20), which lackfunctional E-cadherin-mediated adhesion, expression of other cad-herins is observed. Considering that thede novoexpression of N-cadherin and cadherin-11 may distinguish changes in cell-adhesionprocesses that are important for the invasive and metastatic capacityof the prostate cancer cells, we decided to investigate the expressionof N-cadherin and cadherin-11 in human prostate cancer specimens bysingle- and two-parameter immunofluorescence. By analyzing theprotein expression patterns of these cadherins and correlating them tothe expression patterns of E-cadherin ora-catenin, we aimed atestablishing the pathological role of induced expression of N-cadherinand cadherin-11 in prostate cancer progression.
MATERIALS AND METHODS
Surgical Specimens.On radical prostatectomy or transurethral resection,prostatic adenocarcinoma specimens were snap-frozen in liquid nitrogen. Non-malignant prostate specimens were obtained from cystoprostatectomy. Metas-tases were obtained from operation (pelvic lymph nodes dissection). Fourmmserial sections were used for immunohistochemical analysis. One of the serialsections was stained with H&E to determine the histopathological gradingusing the Gleason score (21).
Immunofluorescence.Sections were fixed using paraformaldehyde (3%)for E-cadherin, N-cadherin, cadherin-11, anda-catenin staining. On preincu-bation with normal sheep serum (diluted 1:20 in PBS) for 30 min.; the sectionswere incubated for 1 h with the primary antibody. Next, the sections wereincubated with biotin-labeled secondary antibody, followed by a streptavidin-fluorescein detection step (Amersham). Antibodies against E-cadherin[HECD-1 (mouse monoclonal antibody, Takara)] and N-cadherin [GC4(mouse monoclonal antibody, Sigma)], an N-cadherin rabbit polyclonal anti-body (Takara), cadherin-11 mouse monoclonal antibody (Ref. 17, cloneNo.16a), and a rabbit polyclonal antibody againsta-catenin, C2081 (Sigma),were used at a dilution of 1:100, 1:30, 1:30, 1:100, and 1:1000, respectively.
Specificity of the antibodies against N-cadherin and Cadherin-11 was de-termined by the comparison of steady-state mRNA levels assessed by Northernanalysis with immunofluorescence, using a panel of cell lines (17). There wasa full correlation between mRNA and immunofluorescence results. Moreover,Western analysis using these antibodies identified proteins of the expectedMW (Mr 135,000 for N-cadherin (17) andMr 120,000 for Cadherin-11.
N-cadherin, cadherin-11, and E-cadherin staining using the mouse mono-clonal antibodies was performed for all of the samples and scored by threeindependent observers (K. T., A. v. B., and E. T. G. R.). E-cadherin was scoredas either normal (membrane staining) or aberrant (negative or heterogeneousstaining) as previously described by Schipperet al. (22) and Umbaset al. (23).N-cadherin staining was judged negative when,5% of the prostate cells werepositive for N-cadherin, or when no significant N-cadherin staining wasobserved. N-cadherin staining was considered positive when 61 5% of theprostate cancer cells showed expression. Furthermore, the staining pattern wasscored,i.e.,membranous, or dotted. Cadherin-11 staining was scored negativewhen weak diffuse staining was observed, particularly in the stroma surround-ing cancer cells, or when no significant staining was observed. Cadherin-11staining was considered positive when 61 5% of the prostate cancer cellsshowed expression, typically in a dotted pattern.
Received 11/19/99; accepted 4/27/00.The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.
1 To whom requests for reprints should be addressed, at Urology Research Laboratory,University Hospital Nijmegen, P. O. Box 9101, 6500 HB Nijmegen, the Netherlands.Phone: 31-24-3614146; Fax: 31-24-3541222; E-mail: [email protected].
Double staining, using FITC- and TRITC2-labeled secondary antibodieswere performed to investigate relative localization of E-cadherin and N-cadherin (rabbit polyclonal) anda-catenin and cadherin-11. The samples werevisualized by either standard epiluminescence fluorescence microscopy (Zeiss,axioscop)using the appropriate filter combinations or CSLM (Leitz DMIRBE,Leica).
Statistical Analyses.The x2 test was used to determine the correlationbetween staining and Gleason score. The odds ratio was used to determine thecorrelation of N-cadherin or cadherin-11 with E-cadherin.
RESULTS
Expression Pattern of N-Cadherin and Cadherin-11 in HumanProstate Cancer Specimens.Using degenerate PCR cloning strate-gies, we have recently identified several cadherins to be induced inprostate cancer cell lines that lack membranous E-cadherin/a-catenincomplexes (17). In particular, N-cadherin and cadherin-11, the ex-pression of which was confirmed by Western blot analysis, seemed tobe of potential relevance for prostate cancer progression. To gaininsight into the role of N-cadherin and cadherin-11 in human prostatecancer progression, we used immunofluorescence analysis to studythe expression patterns of these cadherins in human primary andmetastatic prostate cancer.
In nonmalignant preexisting acini, we never found expression of
N-cadherin,i.e., none of the 12 samples analyzed showed positivestaining. For cadherin-11, we occasionally found a very weak diffusestaining in the stromal cells, which is in agreement with the putativemesenchymal nature of cadherin-11 (13).
In cancer specimens, particularly high-grade (Gleason score:.7; seenext section on “Increased Expression . . . ”), wefound expression ofN-cadherin in the carcinoma cells (Fig. 1,A, D, andG). In the primarycancer specimens, the expression pattern is heterogeneous, with a pre-dominant expression at the membrane (Fig. 1,A andD). Interestingly, inthe high-grade tumors, N-cadherin was expressed more homogeneously,and, moreover, the pattern of staining changed from a membranoushoneycomb-like staining to a dotted pattern (Fig. 1D). Because weperformed double-label immunofluorescence (FITC for N-cadherin andTRITC for E-cadherin), we were able to study the expression of bothcadherins simultaneously. On comparison of the individual fluorescentdyes, it became evident that, in the heterogeneously staining primarytumors, the pattern of staining was complementary,i.e., cells showedeither a predominant expression of E-cadherin or N-cadherin at themembrane (Fig. 1A versus1B, and Fig. 1D versus1E). In the undiffer-entiated tumors (Gleason score: 9, 10) and metastases, E-cadherin wasmostly negative; and all of the cancer cells were positive for N-cadherinalbeit in two patterns, a membranous and a dotted one (Fig. 1,G, H, andI). The complementary staining pattern is best illustrated in the doubleexposures of N-cadherin and E-cadherin-associated immunofluorescence(Fig. 1,C andF).
2 The abbreviations used are: TRITC, Texas Red isothiocyanate; CSLM, confocalscanning laser microscopy.
Fig. 1. Indirect immunofluorescence analysis of expression of N-cadherin (FITC-labeled secondary antibody;A, D, andG); E-cadherin (TRITC-labeled secondary antibody;B, E,and H); and double-exposure of staining for both E- and N-cadherin in prostate cancer specimens, of low-grade (A–C), high-grade (D–F), and a metastatic lesion (G–I). Originalmagnification,3400. Grading was performed by a board-certified pathologist and was based on H&E- stained sections (data not shown). N-cadherin is expressed in a dotted patternand/or at the membrane (A,D). E-cadherin expression is reduced or absent in the cells that are N-cadherin-positive (B, E). In the metastatic lesion, N-cadherin shows homogeneouspositive staining at the membrane (G), but E-cadherin does not (H). Double staining of E- and N-cadherin shows complementary patterns of expression in cancer areas (C,F) and showsonly N-cadherin expression at the membrane (I).
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CADHERIN SWITCHING IN HUMAN PROSTATE CANCER PROGRESSION
These results are in agreement with a switch from E-cadherin expres-sion to N-cadherin expression in the progression of the malignant proc-ess, and one might, therefore, expect intermediate patterns in which thisputative transition is evident. The standard epiluminescence fluorescencemicroscopical evaluation occasionally revealed patterns that were sug-gestive for such intermediate processes, but we considered the judgementof these subtle changes equivocal. Hence, we performed CSLM analysisof the double-label immunofluorescence. Now we could clearly see thatin some areas a transition from E-cadherin to N-cadherin was evident. InFig. 2A, we see—besides E-cadherin- and N-cadherin-positive cell clus-ters—cell-cell contacts where E-cadherin staining and N-cadherin stain-ing are juxtaposed (arrowheads),i.e., not colocalized. In this case, thepattern of N-cadherin staining is similar to that for E-cadherin. However,occasionally we found processes in which E-cadherin and N-cadherinwere expressed in the same cell, albeit in different structures (Fig. 2B).This figure also clearly illustrates that the cells that do express N-cadherinhave a marked decrease in E-cadherin immunoreactivity.
The cadherin-11 immunoreactivity was completely different from thatof N-cadherin. In nonmalignant areas, only weak staining was observedin the stroma (Fig. 3A). Interestingly, the expression of cadherin-11 wasstrongly induced in cancer areas with a typical dotted pattern (Fig. 3B),which was very similar to that observed for N-cadherin. In high-gradecancers and metastases, the expression was no longer confined to thestromal cells but was clearly observed in the cancer cells (Fig. 3,C andD). Sometimes the expression seemed to be at the stromal-epithelialinterface, but coexpression of E-cadherin/a-catenin and cadherin-11 wasalso found in the typical dotted pattern. In the cells coexpressing E-cad-herin/a-catenin and cadherin-11, thede novoexpression of cadherin-11was associated with a decreased expression of E-cadherin.
Increased Expression of N-Cadherin and Cadherin-11 in High-Grade Prostate Cancer and Prostate Cancer Metastases.On thebasis of the detailed analysis of expression patterns of N-cadherin andcadherin-11, we decided to score the samples as follows. N-cadherin waseither negative or positive, whereby we discriminated between membra-nous expression and the dotted pattern in the carcinoma cells. For cad-herin-11, we scored the dotted pattern differentially between stroma and
epithelium,i.e.,a dotted stromal pattern or a dotted stromal1 epithelialpattern. When we confronted the immunofluorescence analysis with thehistopathological data, it became clear that there was a clear correlationwith the Gleason score (Table 1). The membranous expression of N-cadherin increased from 5% of low-grade cases (Gleason score: 4–6) to50% in intermediate- and high-grade cases (Gleason score: 7 and 8–10,respectively). This correlation was statistically significant, as was thecorrelation with decreased expression of E- cadherin (Table 2). In otherwords, there was a significant correlation between decreased expressionof E-cadherin and increased expression of N-cadherin. Moreover, therewas a trend toward a change of localization of the cadherin complexesfrom a typical pattern associated with the adhesion belt to a dottedpattern. We found the highest percentage of tumor cells expressingN-cadherin in the dotted pattern in the metastases (Table 1).
Cadherin-11, is a so-called mesenchymal-type cadherin, and, indeed,we found very weak expression in stroma surrounding nonmalignantsecretory acini. In all cancers, there was a markedly increased expressionof cadherin-11, and of particular interest was the change in the cellularorigin of the expression. In the low-grade tumors, expression was con-fined to the stromal cells, whereas in the intermediate- and high-gradetumors, the carcinoma cells also expressed cadherin-11. The inducedexpression of cadherin-11 in the cancer cells was significantly correlatedwith an increasing Gleason score (Table 3). Moreover, in 9 (60%) of 15of the metastatic lesions, (homogeneous) expression of cadherin-11 in thetypical dotted pattern was also found. As is illustrated by Table 4, themost commonly observed pattern is that of mutual exclusiveness: whenE-cadherin is expressed, there is no expression of cadherin-11 in thecarcinoma cells, and when the E-cadherin staining pattern is aberrant,cadherin-11 is expressed in a dotted pattern in the cancer cells.
DISCUSSION
More than 90% of all malignant tumors are carcinomas and, thus, ofepithelial origin. Interestingly, late in tumor progression, carcinoma cellslose the epithelial differentiation and acquire a more spindle-shaped/mesenchymal morphology, a phenomenon that has been suggested to
Fig. 2. CSLM analysis of the double-label immunofluorescence for N-cadherin (FITC) and E-cadherin (TRITC). Original magnification,3630. CSLM shows besides E-cadherin-and N-cadherin-positive cell clusters, cell-cell contacts where E-cadherin staining and N-cadherin staining are juxtaposed (arrowheads),i.e.,not colocalized (A). CSLM shows processesin which E-cadherin and N-cadherin are expressed in the same cell, albeit in different structures (B). This figure also clearly illustrates that the cells that do express N-cadherin havea marked decrease in E-cadherin immunoreactivity (in the periphery of the process).
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CADHERIN SWITCHING IN HUMAN PROSTATE CANCER PROGRESSION
correlate with metastatic potential (14). In carcinomas, loss or down-regulation of E-cadherin expression is often observed, which correlateswith the degree of malignancy of the tumor (12). Moreover, mechanismsthat interfere with E-cadherin-mediated cell adhesion (e.g.,changes af-fecting the catenins) have been suggested to be involved in epithelial-mesenchymal transition and in increased cellular motility.
Recently, the first reports appeared that described inappropriateexpression of nonepithelial cadherins by epithelial cells as function-ally related to disrupted cell-cell adhesion. A human squamous car-cinoma cell line that displayed a scattered fibroblastic phenotype wasfound to express N-cadherin along with a decreased expression of E-and P-cadherin (24). In addition, in several breast cancer cell lines thatshowed a reduced expression of E-cadherin, high levels of N-cadherinexpression were found (15). Temporary, up-regulation of expressionof N-cadherin has also been suggested to play an important role in themigration of melanoma cells through the endothelium (16, 25). Forprostate cancer, we previously found that cell lines that lack functionalE-cadherin complexes do express N-cadherin and cadherin-11 (17). Inthis report, for the first time, we describe thede novoexpression ofN-cadherin and cadherin-11 using two parameter immunofluores-cence. N-cadherin was expressed in high-grade human prostate can-cers, whereas no expression was found in normal prostatic tissue. Thetechnique used,i.e., double-label immunofluorescence, allowed us toassess the expression pattern of N-cadherin and E-cadherin simulta-neously. In most cases, expression of N-cadherin was observed intumor cells that showed reduced or no staining for E-cadherin; hence,the expression seemed to be mutually exclusive. Further detailedanalysis by CSLM showed that “intermediate” patterns were occa-
sionally observed putatively representing the transition from E-cadherin to N-cadherin expression. These analyses, furthermore, il-lustrated a change in pattern of N-cadherin staining, which suggesteda change in the adherens junction mediated by these cadherins.
The typical honeycomb pattern associated with localization in abelt-like structure disappeared, and a dotted pattern emerged. Thispattern is remarkably similar to that observed for cadherin-11 in thestromal cells, and it is tempting to speculate that this dotted patternrepresents an as yet poorly characterized junctional complex mediat-ing interaction between mesenchymal cells.
Table 1 N-cadherin expression in prostate cancer
n
N-cadherin expression
Negative Dotted Membrane1 dotted
All patients 83 33 22 (26.5%) 28 (33.7%)Gleason score
Table 2 Correlation of N-cadherin expression with E-cadherin expression
E-cadherinexpression n
N-cadherin expression
Negative Dotted Membrane1 dotted
Normal 45 26 10 9 x2 5 14.20Aberrant 38 7 12 19 P 5 0.0008
Fig. 3. CSLM analysis of the double-label im-munofluorescence for cadherin-11 (FITC) anda-catenin (TRITC). Original magnification,3630.Normal prostate specimen (A), primary prostatecancer (B), and lymph node metastases (C,D). Innonmalignant areas, only weak staining is observedin the stroma (A). The expression of cadherin-11 isstrongly induced in stromal areas with a typicaldotted pattern (arrowhead,B). In high-grade can-cers and metastases, the expression was no longerconfined to the stromal cells but clearly observed inthe cancer cells (CandD, arrowheads).a-cateninshows aberrant expression.
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CADHERIN SWITCHING IN HUMAN PROSTATE CANCER PROGRESSION
Moreover, cadherin-11 has been suggested to be up-regulated dur-ing tumor progression: it may play a role in stromal-epithelial inter-actions and, thus, may be involved in migration. This would be inagreement with the interaction of stromal and cancer cells in a subtypeof gastric cancer that is characterized by overexpression of cad-herin-11 (26). A role for cadherin-11 in tumor cell invasion andmetastasis is, furthermore, suggested for breast cancer: in most inva-sive breast cancer cell lines, expression of cadherin-11 is observed,whereas no expression can be detected in any noninvasive cell line(27). In prostate cancer cell lines, expression of cadherin-11 is foundin those lines that have lost functional expression of E-cadherin,attributable to loss of expression ofa-catenin (17).
Double-label immunofluorescence analysis of cadherin-11 expressionrevealed no expression of cadherin-11 in nonmalignant prostate tissue.However, all of the prostate cancer specimens did show a dotted stainingpattern in the stromal cells surrounding the tumor cells and at the interfaceof stroma and tumor cells. Further detailed analysis of the expression ofcadherin-11 provided unequivocal evidence that in high-grade cancers,the tumor cells also express cadherin-11. Even more interesting is thehomogeneous expression of cadherin-11 in the prostate metastatic le-sions. The striking resemblance of the pattern of N-cadherin and cad-herin-11 staining,i.e., in a dotted pattern, indicates that the switch incadherin expression and concomitant change of pattern is, in fact, relatedto the functional interaction between cancer and stromal cells. This, inturn, could be an essential step in the metastatic cascade. Clearly, we canat this point only speculate about the biological functional relation of thecadherin switch and increased metastatic propensity. However, the sig-nificant correlation with histological grade together with the fact that wefind enrichment of this effect in the metastases (i.e., the metastases showa rather homogeneous pattern for both cadherin-11 and N-cadherin ex-pression) corroborates the hypothesis that N-cadherin/cadherin-11-positive cells have a selective advantage to metastasize. It is noteworthy,however, that up-regulation of E-cadherin in metastases has also beenreported (1, 28), which in fact could mean that cadherin switching is adynamic process. The functional role of reexpression of E-cadherin is yetpoorly understood. Cadherin-11, also termed osteoblast(OB-)cadherin isexpressed at high levels in osteoblasts, which makes it, furthermore,tempting to speculate that there is a potential role of cadherin-11 inhoming toward secondary sites, particularly bone.
We, therefore, conclude that we for the first time were able to provideevidence of cadherin switching in advanced human prostate cancer. Thechange in pattern—resembling the pattern of cadherin expression instromal cells—suggests a functional role of homotypic cadherin-medi-ated interactions, in the later stages of cancer progression.
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