The epithelial cells of the prostate are categorized into three types; the basal cells, the luminal epithelial cells and the secretory cells. The basal epithelial cells are the first to appear during normal human prostate development. These cells subsequently undergo differentiation into intermediate and secretory cells. The epithelial cells however, become less differentiated during prostate carcinoma progression. Cells appear with features of both basal and luminal cells (Knudsen and Miranti 2006). The epithelial cells in the prostate gland are bound to one another as well as to the extracellular matrix (ECM). In the normal human prostate, the basal cells are the only cells of the prostate gland attaching to the substratum. However, less differentiated cells infiltrate into the basal cell layer in invasive carcinoma, indicating a change in cell adhesion.
Cell–cell adhesion is mediated by different junction protein complexes, while binding to the ECM is mainly regulated by integrins. The loss of cell–cell adhesion together with the gain of cell–ECM interactions, either transient or permanent, is an indispensable process when the carcinoma progresses to an invasive state (Stewart et al. 2004). Proteins involved in altered cellular adhesion properties may, therefore, function as biological markers.
Cell–cell adhesion is mediated by adherens and tight junctions between epithelial cells. During the metastatic process of epithelial cells, both the composition of these cell junctions and the number of junctions change. Cell adhesion molecules (CAMs), like E-cadherin and N-cadherin as well as β-catenin, are proteins, which are structurally involved in those junctions.
E-cadherin, which is a member of the cadherin family of CAMs, mediates lateral cell–cell adhesion in secretory tissues, like the prostate. E-cadherin is a type-1 Ca2+-dependent cell adhesion molecule and is a major component of adherens junctions in epithelial cells. E-cadherin is located at the cell membrane. It facilitates the binding to different catenins (α-, β- and γ-catenin) that associate with actin filaments and the actin cytoskeleton within the cell.
The expression of E-cadherin has been extensively studied using tissue micro array (TMA) in prostate tumor, derived from radical prostatectomy (RP) (Rubin et al. 2001). In low-grade carcinomas with negative surgical margin, benign and normal tissue E-cadherin was normally expressed. The expression of E-cadherin was decreased in higher grade carcinomas and carcinomas with a positive surgical margin. Furthermore, metastatic tissues showed strong E-cadherin staining. This is rather controversial, as the related primary tumors showed loss of E-cadherin expression. The re-expression of E-cadherin in metastasis may imply that the loss of E-cadherin is a transient event occurring during invasion and diapedesis. The re-expression of E-cadherin in metastatic tissue may be regulated by paracrine signaling from cells of the metastatic environment.
The low expression of E-cadherin has been correlated with prostate specific antigen recurrence in serum after RP, suggesting that aberrant E-cadherin expression could be predictive of clinical outcome (Rhodes et al. 2003). The expression of E-cadherin is inversely correlated to tumor grade, even within one RP specimen containing both benign and malignant tissues (Jaggi et al. 2005). This also underlines the difficulty of E-cadherin expression analysis using prostate biopsies compared to PC specimens. Most prostate tumors are multi-focal and heterogeneous. During sampling of prostate biopsies, foci of malignant carcinoma within the prostate could be missed.
Most studies used a large number of samples (ranging from 16 to 259) to analyze the correlation of E-cadherin expression and tumor grade. Although a difference could be seen between the two groups of samples (high- vs. low Gleason score), not all individual cases showed a low E-cadherin expression in combination with a high Gleason score, indicating that E-cadherin expression may not be used as an individual marker. However, we may use E-cadherin expression data in combination with other markers of metastasis.
Besides decreased expression of E-cadherin, various studies were directed to the role of E-cadherin gene (CDH1) polymorphisms (Bonilla et al. 2006; Li et al. 2006; Verhage et al. 2002). A -160 C/A single nucleotide polymorphism was found in allele A. (Liet al. 2006). This polymorphism showed approximately 70% decreased transcription of allele A. This may be a cause of the decreased E-cadherin expression as observed in prostate carcinoma. The suitability of this polymorphism in predicting prostate carcinoma metastasis is, however, controversial. An elevated risk for prostate carcinoma was observed in allele A carriers in a Dutch population, with a higher risk for sporadic cancers (approximately fivefold) compared to hereditary cancer (approximately twofold) (Verhage et al. 2002). This is contradictory to Swedish results, which showed a higher risk for hereditary cancers (Jonsson et al. 2004). More CDH1 polymorphisms were studied in African-Americans, Jamaicans and European-American men (Bonilla et al. 2006). This study showed that a combination of polymorphisms (160A and +54T) might present a susceptibility for prostate cancer in European populations.
The discrepancy between results of various studies reveals that more research must be done to link CDH1 polymorphisms to metastatic disease in prostate cancer. It is suggested that polymorphisms decrease E-cadherin transcription and thereby its expression. Therefore, E-cadherin protein expression itself, instead of gene polymorphisms, may be a valid marker. It could be even more promising to use E-cadherin protein expression data in combination with other markers.
N-cadherin is just like E-cadherin a type-I cadherin. While E-cadherin is mostly expressed in epithelial cells, N-cadherin is expressed in various cell types including nerve, myocardial and mesenchymal cells. Increase in N-cadherin expression is one of the features of epithelial to mesenchymal transition (EMT), together with loss of E-cadherin expression. The expression of E-cadherin declines, while N-cadherin expression increases during the process of EMT. N-cadherin facilitates a more dynamic cell–cell adhesion. N-cadherin expression in prostate cancer is less well studied compared to E-cadherin expression. In one study, the switch of cadherin expression was associated with higher grade tumors (Jaggi et al. 2006). In a total of 44 PC specimens 45% showed N-cadherin expression. Only 7% of the Gleason 5–6 tumors showed positive expression, compared to 65% of Gleason grade 7, and 57% of Gleason score 8–10. Although N-cadherin expression correlates with Gleason score, no data are available correlating N-cadherin expression to PSA recurrence, metastasis or tumor progression.
β-Catenin is a multifunctional protein; it is not only involved structurally in the adherens junction complex, but it also acts as signaling molecule. It connects the cytoplasmic part of E-cadherin to the cytoskeleton together with α catenin in the adherens junctions. When adherens junction complexes are lost, a soluble form of β-catenin becomes localized in the cytoplasm and some free β-catenin is transported to the nucleus. The cytoplasmic expression of β-catenin is rather low in normal cells as it is easily targeted for ubiquitination by GSK-3β phosphorylation and subsequently degraded by proteosomes. However, signaling by the Wnt pathway represses the degradation of β-catenin by degrading the GSK-3β/β-catenin complex. This leads to suppressed phosphorylation of β-catenin and, consequently, accumulation of the protein. The Wnt signaling might be involved in prostate cancer development (Yardy and Brewster 2005). Besides the Wnt signaling pathway the Pi-3K/Akt pathway also seems to be involved in regulating the soluble pool of free β-catenin. The Pi-3K/Akt pathway signaling is also involved in the development of prostate carcinoma, mainly by loss of the PTEN Akt suppressor (Downward 2004).
The higher levels of soluble β-catenin may lead to higher amounts of this protein in the nucleus were it exerts its transcriptional function. It interacts with different DNA-binding transcription factors and especially the TCF/lymphoid enhancer factor (LEF) family, which includes TCF1, TCF3–4 and LEF1. Those DNA-binding proteins repress the transcription of target genes by binding transcriptional repressors. β-catenin competes for binding with those repressors, leading to transcriptional activation of the target genes. The transcriptional activation of β-catenin (CRT) is involved in numerous processes in normal cells, from embryonic anterior–posterior axis specification to tissue development. Hyper-activation of CRT signaling may elicit pronounced morphology and trans-differentiation in prostatic neoplasia (Chesire et al. 2002). Although the loss of adherens junctions leads to accumulation of free soluble β-catenin it does not necessarily result in higher activation of β-catenin signaling as free β-catenin is rapidly degraded. However, in combination with ubiquitination repression, β-catenin accumulation could be, partially, responsible for prostate carcinoma progression due to elevated CRT signaling.
Normal prostate tissue shows high membranous β-catenin expression and low nuclear β-catenin staining. Prostatic intraepithelial neoplasia (PIN) lesions show less expression of membranous β-catenin. This indicates that the loss of adherens junctions is an event occurring early in tumor development, assuming PIN lesions as precursors of prostate carcinoma.(Jaggi et al. 2005) However, the loss of membranous β-catenin occurs at a very low rate and does not correlate with tumor stage or grade. The loss of membranous β-catenin could be a direct consequence of loss of adherens junctions early in tumor development.
Membranous β-catenin was lowered in both benign prostate hyperplasia (BPH), a non-malignant enlargement of the prostate, and localized prostate cancer (Horvath et al. 2005). Nuclear β-catenin staining is stronger in BPH compared to normal prostate tissue. This indicates that β-catenin accumulation in the cytoplasm, due to loss of membranous β-catenin, leads to higher nuclear levels. However, when compared with localized prostate cancer, BPH had higher levels of nuclear staining. Advanced prostate cancer showed even less nuclear β-catenin staining compared with localized disease. Furthermore, lower levels of nuclear β-catenin correlate with poorer prognosis (Horvath et al. 2005).
β-catenin signaling may both, repress and promote tumor growth. Higher nuclear β-catenin levels indicate tumor repression while lower nuclear β-catenin levels indicate tumor growth. The stabilization of nuclear β-catenin levels is regulated by various cofactors and the balance between those factors and β-catenin results in tumor promotion, or repression. The fact that relatively low nuclear β-catenin levels do correlate with disease outcome suggests nuclear β-catenin as a possible marker. More studies of low-risk prostate cancer may clarify the role of nuclear β-catenin levels during prostate cancer progression.
Cell-ECM adhesion/focal adhesions
Besides cell–cell adhesion, epithelial cells are also connected to the ECM. The interaction with the ECM is indispensable for the traveling of a metastatic cell. The interaction with the ECM changes when the cell has metastatic potential. Integrins may be involved in such changes.
Integrins are important mediators in the attachment of epithelial cells to the ECM. These appear in complexes at the cell surface known as focal adhesions. Integrins play an important role in tumor-associated signaling events besides anchoring to the ECM. Focal adhesion kinase (FAK) is an important mediator of integrin signaling. Integrin-dependent signaling is supposed to affect cell growth, anchorage-dependent differentiation, adhesion, motility and apoptosis.
Integrins appear in heterodimeric structures containing an α and β chain. The predominant structures found on epithelial cells are the α5β1, α6β1 and the α6β4 integrins. The suprabasal and secretory cells are distinct from the basal cells in the expression of integrins and adhesion molecules that are able to connect to the substratum. In normal prostate glands, only the basal cells express integrins connecting them to the substratum. The α6β4 integrin, for example, is an important component of the hemidesmosome expressed at the basal surface in most stratified epithelial cells. The hemidesmosome links the cytoskeleton intermediate filaments to laminin-5 in the ECM.
Basal epithelial cells are lost during PC progression. Partial loss of the basal lamina is a hallmark of high-grade PIN. The basal cell lining almost completely disappears in PC. The expression of integrins changes together with the loss of the basal lining. The β4 integrin, for example, was lost in PIN lesions together with basal cell-lining and in prostate carcinoma the expression of β4 integrins was totally lost (Davis et al. 2001). Although the β4 integrin was lost, the α6β1 integrin showed to be continuously expressed through all cancer stages, which may indicate that the composition of integrins changes during tumor progression.
Expression of laminin-5, the major ligand of α6β4 integrin, is also declining through PC progression (Davis et al. 2001). This may indicate that the composition of the local ECM is rather important for physiology. The continuous expression of the α6β1 integrin together with the loss of α6β4 integrin may reflect the motility of the cancer cells. Loss of the α6β4 integrin may give a less-stable attachment to the ECM, while the continued expression of the α6β1 integrin may provide enough attachment for the cells to become mobile. This may also explain why prostate cancer cells favor to move along laminin-5 coated nerves (Cress et al. 1995).
The composition of integrins in prostate cancer tissue, like the combination of loss of α6β4 and continuous expression of α6β1, may reflect the capability of the cells to metastasize. Such a combination may, therefore, be a valid biological marker.
Focal adhesion kinase
Signaling of integrins is mediated by the connection of integrins to focal adhesion kinase (FAK). FAK, a protein tyrosine kinase, is localized at focal adhesions at the cell membrane and signals through the phosphoidyllinositol 3 kinase (PI 3) pathway. This integrin-dependent signaling is, in general, known to regulate several tumor-progressing processes as cell growth, adhesion-based differentiation, adhesion, motility and apoptosis (Downward 2004). Increasing levels of FAK in human prostate cancer cell lines correlate with greater metastatic potential (Tremblay et al. 1996). FAK was predominantly expressed in the basal epithelial cell layer as shown in an immunohistochemical analysis of various prostate specimens (Rovin et al. 2002). FAK overexpression appeared in PIN lesions, with a clear distribution of high FAK staining in neoplastic cells while normal cells in the surrounding normal tissues did not show elevated expression. Furthermore, benign prostate hyperplasia (BPH) did not show a change in FAK expression compared to normal tissue. Beside the FAK overexpression in PIN lesions, higher grade and metastatic carcinomas retained the elevated FAK expression, suggesting an important role for FAK in tumor progression (Rovinet al. 2002). The elevated expression of FAK in early stage carcinoma may, therefore, be used as a biological marker.