The prognostic value of E-cadherin and the cadherin-associated molecules alpha-, beta-, gamma-catenin and p120ctn in prostate cancer specific survival: a long-term follow-up study.

OBJECTIVES: To determine the value of loss of expression of E-cadherin and cadherin associated molecules as prognostic markers for prostate cancer patients in a long-term follow-up study. METHODS: Sixty-five prostate cancer specimens, obtained from patients with different stages of prostate cancer who underwent a radical prostatectomy or TUR-P between 1987 and 1991, were used for immunohistochemical analysis of the expression pattern of E-cadherin, alpha-, beta-, gamma-catenin and p120(ctn). Clinical records of these patients were studied for follow-up data and the prognostic value of expression of these adhesion molecules was determined by Kaplan-Meier survival analyses and multivariable proportional hazard regression analysis. RESULTS: Normal staining patterns were found in 36 cases (55.4%) for E-cadherin, 37 cases (56.9%) for alpha-catenin, 40 cases (61.5%) for beta-catenin, 25 cases (38.5%) for gamma-catenin, and 40 cases (61.5%) for p120(ctn). Overall, a strong correlation was found between the expression of E-cadherin and other cadherin-associated molecules. The 5-year survival rates for each staining were as follows: E-cadherin (normal 79.2%, aberrant 26.8%), alpha-catenin (normal 79.2%, aberrant 26.8%), beta-catenin (normal 73.1%, aberrant 27.3%), gamma-catenin (normal 86.4%, aberrant 37.1%), and p120(ctn) (normal 72.8%, aberrant 30.0%). There was a significant difference in survival between normal and aberrant expression in each staining (log rank P < 0.0001). The proportional hazard regression model including tumor stage and Gleason score revealed alpha-catenin expression as the best prognostic marker for patients with prostate cancer. CONCLUSIONS: Our data revealed a strong correlation between E-cadherin expression and other cadherin-associated molecules. Among these markers, alpha-catenin seems the best prognostic marker for prostate cancer specific survival. Larger studies are needed to confirm this result.

Isolation and characterization of the promoter of the human prostate cancer-specific DD3 gene.

Recently, we have described a novel gene, DD3, which is one of the most prostate cancer-specific genes described to date (Bussemakers, M. J. G., van Bokhoven, A., Verhaegh, G. W., Smit, F. P., Karthaus, H. F. M., Schalken, J. A., Debruyne, F. M. J., Ru, N., and Isaacs, W. B. (1999) Cancer Res. 59, 5975-5979). The prostate cancer-specific expression of DD3 indicates that the DD3 gene promoter is a promising tool for the treatment of prostate cancer. To identify the promoter elements that are responsible for the prostate cancer-specific expression of DD3, we have isolated and characterized the DD3 promoter. Sequence analysis of the DD3 5'-flanking region was performed and several promoter-human growth hormone reporter constructs were prepared, which were transiently transfected in the DD3-positive cell line LNCaP and several DD3-negative cell lines. Using a 500-base pair DD3 promoter construct, we could detect promoter activity in LNCaP cells, which was not affected by increasing the size of the constructs. Truncated constructs, however, showed an increased transcriptional activity, suggesting the presence of a silencer that negatively regulates the expression of DD3. DNase-I footprint analysis, using nuclear extracts from LNCaP cells, revealed the presence of three DNase-I-protected areas within the DD3 proximal promoter. We show that the high mobility group I(Y) protein binds to one of the DNase-I-protected areas and recruits another, yet unidentified, protein to the DD3 promoter in LNCaP cells.

Silencing of CD44 expression in prostate cancer by hypermethylation of the CD44 promoter region.

Loss of the CD44 transmembrane glycoprotein in primary prostate cancer has been shown to be associated with unfavorable clinical behavior. Moreover, the majority of prostate cancer metastases lack expression of this molecule. The mechanism of CD44 silencing in prostate cancer was investigated using both patient material and in vivo-propagated human prostate cancer xenografts. In 9 of 11 lymph node metastases of prostate cancer, we demonstrated by methylation-sensitive restriction enzyme digestion that the promoter region of the CD44 gene is methylated, indicating that this represents a major mechanism of CD44 silencing. Similarly, in 6 out of 12 in vivo-growing human prostate carcinoma xenograft models, hypermethylation of the CD44 gene was found. The extent of CpG island methylation was investigated by nucleotide sequencing after bisulphite modification of the CD44 promoter region. In the xenografts displaying hypermethylation, the examined 14 CpG sites in the CD44 transcription regulatory domain, including a Sp1 binding site, were consistently methylated. This correlated with reduced CD44 expression or lack of CD44 expression at mRNA and protein levels. In the xenografts lacking hypermethylation of the CD44 gene, high levels of CD44 mRNA and protein were expressed in some models, whereas in others CD44 mRNA expression was only detectable by RT-PCR and the CD44 protein could hardly be detected or was not detected at all. The results indicate that, in most prostate cancers, loss of CD44 expression is associated with extensive hypermethylation of the CpG island of the CD44 promoter region, but other, posttranscriptional mechanisms may also lead to CD44 loss.

Cadherin switching in human prostate cancer progression.

The progression of carcinomas is associated with the loss of epithelial morphology and a concomitant acquisition of a more mesenchymal phenotype, which in turn is thought to contribute to the invasive and/or metastatic behavior of the malignant process. Changes in the expression of cadherins, "cadherin switching," plays a critical role during embryogenesis, particularly in morphogenetic processes. Loss of E-cadherin is reported to be associated with a poor prognosis; however, thus far, evidence (R. Umbas, et al., Cancer Res. 54: 3929-3933, 1994) for up-regulation of other cadherins has only been reported in vitro, ie., we have found evidence (M. J. G. Bussemakers et al., Int. J. Cancer, 85: 446-450, 2000) for cadherin switching in prostate cancer cell lines (up-regulation of N-cadherin and cadherin-11, two mesenchymal cadherins, in cell lines that lack a functional E-cadherin-catenin adhesion complex). Here, we report on the immunohistochemical analysis of the expression of N-cadherin and cadherin-11 in human prostate cancer specimens. N-cadherin was not expressed 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. Cadherin-11 is expressed in the stroma of all prostatic tumors, in the area where stromal and epithelial cells are found. In addition, cadherin-11 is also expressed in a dotted pattern or at the membrane of the epithelial cells of high-grade cancers. In a number of metastatic lesions, N-cadherin and cadherin-11 are expressed homogeneously. These data raise the possibility that cadherin switching plays an important role in prostate cancer metastasis.

Polymorphisms in the vitamin D receptor gene and the androgen receptor gene and the risk of benign prostatic hyperplasia.

OBJECTIVE: Little is known about risk factors for the development of benign prostatic hyperplasia (BPH). Recently, associations were observed between prostate cancer (CaP) risk and polymorphisms in the vitamin D receptor (VDR) gene and the androgen receptor (AR) gene. Since both receptors are relevant for prostate growth, the VDR and AR are also expected to be involved in the development of BPH. The objective of this study is to establish the relationship between the risk of BPH and a polymorphism in the number of CAG repeats in the AR gene and a TaqI restriction enzyme polymorphism in the VDR gene. METHODS: For this study, 98 patients who had been treated for BPH-related complaints and 61 convenience controls (predominantly bladder cancer patients) were recruited from the outpatient clinic. DNA was isolated from peripheral blood, and genotyping was performed with PCR-based methods. Means as well as odds ratios (ORs) with 95% confidence intervals (CI) were calculated using SPSS software. RESULTS: The mean number of CAG repeats in the AR gene in patients and controls was found to be similar: 21.8 (SD = 2.8) and 21.9 (SD = 2.9), respectively. In the subgroup of patients with a prostate volume of at least 50 cm(3), the mean number of repeats was 21.5 (SD = 2.6). The OR for BPH for individuals with homozygous presence of the VDR TaqI restriction fragment length polymorphism (RFLP) (tt) versus individuals with homozygous absence (TT) or heterozygotes (Tt) was found to be 1.0 (95% CI 0.4-2.4). For individuals with a prostate volume of at least 50 cm(3), the OR was 1.2 (95% CI 0.5-3. 2). CONCLUSION: Unlike earlier observations in prostate cancer, we did not find an association between the CAG repeat polymorphism in the AR gene and the TaqI RFLP polymorphism in the VDR gene and the risk of BPH.

Complex cadherin expression in human prostate cancer cells.

Changes in cell-cell interactions are critical in the process of cancer progression. Likewise, it has been shown that loss of expression of the cell adhesion molecule E-cadherin is associated with grade, stage, and prognosis in many carcinomas, including prostate cancer. Impaired E-cadherin-mediated interactions result in an invasive phenotype; however, the mere loss of cell-cell contact and communication is not the sole explanation for the observed correlation between loss of E-cadherin-mediated adhesion and poor clinical outcome. Using a degenerate cloning strategy for sequences that are highly conserved between the various cadherins, we found several other cadherins (N- and P-cadherin and cadherin-4, -6, and -11) to be expressed in human prostate cancer cells. Our data suggest that besides loss of E-cadherin function, also (upregulation of) expression of other cadherins is involved in the acquisition of an invasive and/or metastatic phenotype. Especially, changes in the expression of N-cadherin and cadherin-11 may play an important role in prostate cancer progression.

Coordinate recruitment of E-cadherin and ALCAM to cell-cell contacts by alpha-catenin.

Here we report on the role of alpha-catenin in the cellular localization of activated leukocyte cell adhesion molecule, ALCAM, and cadherin-mediated cell adhesion in human prostate cancer cells. Cell lines that have a functional E-cadherin-mediated cell adhesion (DU-145 and LNCaP) show ALCAM staining at cell-cell contacts. In contrast, in cell lines that lack alpha-catenin expression (ALVA-31, PC-3, and PPC-1), E-cadherin-mediated adhesion is disturbed and ALCAM staining is cytoplasmic. A role of alpha-catenin in the recruitment of E-cadherin and ALCAM to cell-cell contacts was established by transfection of an alpha-N-catenin construct into cell lines ALVA-31 and PC-3. This resulted not only in the correct assembly of E-cadherin/alpha-catenin complexes at the cell membrane but also in localization of ALCAM to cell-cell contacts, indicating that indeed alpha-catenin affects ALCAM localization.

DD3: a new prostate-specific gene, highly overexpressed in prostate cancer.

Prostate cancer is the most commonly diagnosed malignancy and the second leading cause of cancer-related deaths in the Western male population. Despite the tremendous efforts that have been made to improve the early detection of this disease and to design new treatment modalities, there is still an urgent need for new markers and therapeutic targets for the management of prostate cancer patients. Using differential display analysis to compare the mRNA expression patterns of normal versus tumor tissue of the human prostate, we identified a cDNA, DD3, which is highly overexpressed in 53 of 56 prostatic tumors in comparison to nonneoplastic prostatic tissue of the same patients. Reverse transcription-PCR analysis using DD3-specific primers indicated that the expression of DD3 is very prostate specific because no product could be amplified in 18 different normal human tissues studied. Also, in a sampling of other tumor types and a large number of cell lines, no expression of DD3 could be detected. Molecular characterization of the DD3 transcription unit revealed that alternative splicing and alternative polyadenylation occur. The fact that no extensive open reading frame could be found suggests that DD3 may function as a noncoding RNA. The DD3 gene was mapped to chromosome 9q21-22, and no homology of DD3 to any gene present in the computer databases was found. Our data indicate that DD3 is one of the most prostate cancer-specific genes yet described, and this makes DD3 a promising marker for the early diagnosis of prostate cancer and provides a powerful tool for the development of new treatment strategies for prostate cancer patients.

Molecular analysis of multifocal prostate cancer lesions.

To analyse the origin of multifocal prostate cancer lesions, radical prostatectomy specimens from 17 patients were examined. As a marker of genetic lineage, the allelotype based on 33 microsatellite loci was compared between the different tumours present in a given case. Some results provide evidence suggestive of a clonal origin of multiple tumours in a subset of the prostates. In five cases, for example, comparison of multifocal tumour lesions within a given case revealed at least two concordant changes in allelic imbalance (AI) sequence dosages at different loci. In addition, considerable heterogeneity of allelotype was found within and among tumour foci of a given case. In five of the six tumours analysed for intratumour heterogeneity, for example, more than five discordant AI changes were found in one tumour region but not in the other. Conclusions regarding the clonality of such heterogeneous lesions are difficult to draw. A high frequency of AI changes in four lesions exhibiting prostatic intraepithelial neoplasia (mean 6.5 changes per lesion, range 3-6) was found, compared with eight primary tumours present in the same cases (mean 5.8 changes per lesion, range 3-6). The interpretation of AI associated with clinically detected prostate cancer remains a highly complex issue. The fact that no clear evidence was obtained for either a clonal or a non-clonal origin of multiple lesions in a given prostate indicates that several different mechanisms are likely to operate in establishing the allelotype and that additional evidence from unique mutations or selective gene inactivation may be necessary to obtain definitive results.

Changes in gene expression and targets for therapy.

A better understanding of the molecular changes associated with the onset and progression of prostate cancer may provide us with a rational basis for the development of new diagnostic and therapeutic tools. Likewise, the recent identification of critical biochemical pathways, including angiogenesis, programmed cell death, cell adhesion and signal transduction, provide us with promising targets for therapeutic approaches. Furthermore, the identification and characterization of new tumor-specific antigens or prostate-cancer-specific gene promoters could be instrumental for the development of new treatment modalities. Many research groups are trying to identify genes that are involved in prostate cancer development and which may serve as new tumor markers and potential targets for therapy. In addition to prostate-specific antigen, prostate-specific membrane antigen and human kallikrein-2, the recently identified prostate stem cell antigen may also provide us with a new tool for the diagnosis and treatment of prostate cancer. Our own studies led to the identification of DD3, a gene that is strongly overexpressed in human prostatic cancers and the expression of which appears to be restricted to the prostate. Further studies are necessary to establish the clinical usefulness of these new prostate-cancer-specific genes for the management of prostate cancer patients.

Cadherin-11 is expressed in invasive breast cancer cell lines.

In several cancers, including breast cancer, loss of E-cadherin expression is correlated with a loss of the epithelial phenotype and with a gain of invasiveness. Cells that have lost E-cadherin expression are either poorly invasive with a rounded phenotype, or highly invasive, with a mesenchymal phenotype. Most cells lacking E-cadherin still retain weak calcium-dependent adhesion, indicating the presence of another cadherin family member. We have now examined the expression of the mesenchymal cadherin, cadherin-11, in breast cancer cell lines. Cadherin-11 mRNA and protein, as well as a variant form, are expressed in the most invasive cell lines but not in any of the noninvasive cell lines. Cadherin-11 is localized to a detergent-soluble pool and is associated with both alpha- and beta-catenin. Immunocytochemistry shows that cadherin-11 is localized to the cell membrane at sites of cell-cell contact as well as at lamellipodia-like projections, which do not interact with other cells. These results suggest that cadherin-11 expression may be well correlated with the invasive phenotype in cancer cells and may serve as a molecular marker for the more aggressive, invasive subset of tumors. Cadherin-11 may mediate the interaction between malignant tumor cells and other cell types that normally express cadherin-11, such as stromal cells or osteoblasts or perhaps even with the surrounding extracellular matrix, thus facilitating tumor cell invasion and metastasis.

Molecular cloning of an alternative human alphaE-catenin cDNA.

The cytoplasmic protein alpha-catenin plays a crucial role in E-cadherin mediated cell-cell adhesion by binding E-cadherin to the cytoskeleton via beta- or gamma-catenin and actin. Functional loss of one of these interacting components leads to decreased cell-cell adhesion, and therefore to loss of epithelial integrity. Northern analysis revealed two distinct alphaE-catenin transcripts in different cell lines, whereas apparently only one protein is expressed. Because of the biological importance of this protein we sought to molecularly characterize the differences between the two observed transcripts. cDNA cloning and sequence analysis revealed the earlier described 3.4 kb alphaE-catenin transcript and an alphaE-catenin transcript of approximately 3.8 kb. This larger transcript contains a 321 bp extension in the 3'UTR sequence, which probably arises as a result of alternative polyadenylation. Considering the presence of AU-rich sequences in the extension, it may be involved in mRNA stability.

The role of cell adhesion molecules and proteases in tumor invasion and metastasis.

Over the past 10 years it has become apparent that invasion and metastasis are extremely complex processes; neoplastic cells must escape from the primary tumor, degrade the extracellular matrix, migrate to distant sites, arrest in the capillaries, and migrate through the basement membrane and underlying connective tissue to the metastatic site. Therefore, tumor cells must exhibit considerable flexibility in their adhesive interactions, and this is reflected in a complex and dynamic expression pattern of cell adhesion molecules, proteases, protease inhibitors, motility factors, and growth factors. Despite the recent explosion of information regarding adhesion-related molecules, questions as to their possible roles in normal tissue architecture and as to how alterations in their expression or structure may be responsible for the progression from a single malignant cell to a lethal metastatic disease need further investigation. Moreover, efforts should be made to use the obtained knowledge to contribute to improvements in the clinical management of cancer. In this review the different classes of cell adhesion molecules and proteases are summarized, with special emphasis being placed on molecules that have been shown to correlate with invasion and metastasis. Furthermore, the role of E-cadherin in cell adhesion and invasive processes is discussed in more detail, since E-cadherin may be considered promising as a candidate among cell-adhesion-regulating molecules to be used as a biomarker for malignancy. We also elaborate on the role of the catenins, which associate with and are important for the functioning of E-cadherin.

Regional loss of imprinting of the insulin-like growth factor II gene occurs in human prostate tissues.

In most tissues, the insulin-like growth factor II gene (IGF-II) demonstrates imprinting, being expressed exclusively from the paternal allele. Recently, a loss of IGF-II imprinting (i.e., biallelic expression) has been found in sporadic Wilm's tumors and lung carcinomas, and this molecular event may contribute to the pathogenesis of these tumors. Here, we report that in prostates removed at radical surgery for localized adenocarcinoma, both the cancer and the associated normal peripheral zone tissue have a pronounced biallelic expression of the IGF-II gene. However, this pattern of gene expression is uncommon in periurethral samples of benign prostatic hyperplasia (BPH) from the same specimens. We analyzed the status of genomic imprinting at the IGF-II locus in prostate specimens removed for carcinoma using an ApaI polymorphism in the 3' untranslated exon of the IGF-II gene. First-strand cDNA synthesis and subsequent PCR amplification were performed on 13 of 35 radical prostatectomy specimens found to be informative for analysis of allele-specific expression. Biallelic expression for IGF-II RNA was demonstrated in 10 (83%) of 12 tumor samples and 8 (73%) of 11 matched peripheral zone prostate samples but in only 2 (18%) of 11 BPH samples. RNA transcripts were readily demonstrated by Northern blot analysis, and differences in expression were not noted among normal, BPH, and tumor prostate tissues. In situ hybridization revealed production of IGF-II by both the epithelium and stroma. The finding of a frequent biallelic expression of IGF-II in peripheral prostate specimens suggests a regional pattern of IGF-II gene regulation exists in prostate tissue. We hypothesize that this tissue-specific pattern of gene expression may participate in the marked predilection of peripheral prostatic tissue for the development of carcinogenesis.

Cloning and characterization of the human invasion suppressor gene E-cadherin (CDH1).

E-cadherin is a Ca(2+)-dependent epithelial cell-cell adhesion molecule. Downregulation of E-cadherin expression often correlates with strong invasive potential and poor prognosis of human carcinomas. By using recombinant lambda phage, cosmid, and P1 phage clones, we isolated the full-length human E-cadherin gene (CDH1). The gene spans a region of approximately 100 kb, and its location on chromosome 16q22.1 was confirmed by FISH analysis. Detailed restriction mapping and partial sequence analysis of the gene allowed us to identify 16 exons and a 65-kb-long intron 2. The intron-exon boundaries are highly conserved in comparison with other "classical cadherins." In intron 1 we identified a 5' high-density CpG island that may be implicated in transcription regulation during embryogenesis and malignancy.

Molecular biology of prostate cancer progression.

A number of genetic changes have been documented in prostate cancer, ranging from allelic loss to point mutations and changes in DNA methylation patterns (summarized in Fig. 1). The most consistent changes seen are those of allelic loss events, with the majority of tumours examined showing loss of alleles from at least one chromosomal arm. The short arm of chromosome 8, followed by the long arm of chromosome 16, seem to be the most frequent regions of loss, suggesting the presence of novel tumour suppressor genes. Deletions of one copy of the RB and TP53 genes are less frequent as are mutations of the TP53 gene, and accumulating evidence suggests the presence of an additional tumour suppressor gene on chromosome 17p, which is frequently inactivated in prostate cancer. Alterations in the E-cadherin/alpha catenin mediated cell-cell adhesion mechanism appear to be present in almost half of all prostate cancers and may be critical to the acquisition of metastatic potential of aggressive prostate cancers. Finally, altered DNA methylation patterns have been found in the majority of prostate cancers examined, suggesting widespread alterations in methylation modulated gene expression. The presence of multiple changes in these tumours is consistent with the multistep nature of the transformation process. Finally, efforts to identify prostate cancer susceptibility loci are under way, which may elucidate critical early events in prostatic carcinogenesis.

Molecular biology of prostate cancer.

A number of genetic changes have been documented in prostate cancer, ranging from allelic loss to point mutations and changes in DNA methylation patterns (summarized in Fig 1). To date, the most consistent changes are those of allelic loss events, with the majority of tumors examined showing loss of alleles from at least one chromosomal arm. The short arm of chromosome 8, followed by the long arm of chromosome 16 appear to be the most frequent regions of loss, suggesting the presence of novel tumor suppressor genes. Deletions of one copy of the Rb and p53 genes are less frequent as are mutations of the p53 gene, and accumulating evidence suggests the presence of an additional tumor suppressor gene on chromosome 17p, which is frequently inactivated in prostate cancer. Alterations in the E-cadherin/alpha catenin mediated cell-cell adhesion mechanism appear to be present in almost half of all prostate cancers, and may be critical to the acquisition of metastatic potential of aggressive prostate cancers. Finally, altered DNA methylation patterns have been found in the majority of prostate cancers examined, suggesting widespread alterations in methylation-modulated gene expression. The presence of multiple changes in these tumors is consistent with the multistep nature of the transformation process. Finally, efforts to identify prostate cancer susceptibility loci are underway and will hopefully elucidate critical early events in prostatic carcinogenesis.

Transcriptional regulation of the human E-cadherin gene in human prostate cancer cell lines: characterization of the human E-cadherin gene promoter.

Decreased expression of the Ca(2+)-dependent cell adhesion molecule E-cadherin is observed in several poorly differentiated carcinomas and is presumably associated with an invasive phenotype of these tumors. Evidence accumulated so far indicates that decreased transcription is a major mechanism leading to defective E-cadherin function. Therefore, we isolated and characterized the human E-cadherin gene promoter and studied the transcriptional regulation of this gene in two human prostate cancer cell lines, one expressing E-cadherin (PC-3), the other one not expressing E-cadherin (TSU-pr1). We show that the E-cadherin promoter is not active in the non-expressing cells and that this may be due to the binding of a repressor protein to the promoter.

The genes for the calcium-dependent cell adhesion molecules P- and E-cadherin are tandemly arranged in the human genome.

Cadherins constitute a gene family of Ca(2+)-dependent cell-cell adhesion molecules involved in the morphogenesis and maintenance of tissue integrity. E- and P-cadherin are members of the cadherin family that are both expressed in epithelial tissues. Here we present the localization of the human P-cadherin gene at 32 kb upstream of the human E-cadherin gene, mapping it to chromosome 16q22.1. Tandem arrangement of two cell-cell adhesion molecules from the cadherin family has also been reported in chicken. This is the first evidence for the direct linkage of two cadherins in mammals. The evolutionary conservation of the tandem arrangement of two genes encoding cell adhesion molecules suggests that the close proximity of the genes may be important for the regulation of the genes.