Background: Changes in junctional catenin expression may compromise cadherin-mediated adhesion, increasing cell malignant properties such as invasive and metastatic abilities. Altered expression of α-, β-, γ- and p120-catenin has been reported to be associated with E-cadherin loss or decreased expression, in both breast carcinomas and breast cancer cell lines.
Aims and Methods: To investigate the expression and subcellular localisation of p120- and β-catenin in a series of human invasive breast carcinomas, and correlate it with biological markers and clinicopathological parameters.
Results: Both catenins frequently exhibited a reduced membranous or cytoplasmic staining pattern. These alterations were significantly correlated with lack of both E-cadherin and oestrogen receptor-α expression. It was possible to associate the expression of β-catenin with histological grade, tumour size and nodal status, suggesting a relevant role for this catenin as a prognostic factor. The majority of E- and P-cadherin co-expressing tumours were related to cytoplasmic expression of p120-catenin; in this group of breast carcinomas, patient survival was poor.
Conclusion: Results indicate that p120-catenin cytoplasmic accumulation may play an important role in mediating the oncogenic effects derived from P-cadherin aberrant expression, including enhanced motility and invasion, particularly in tumours which maintain E-cadherin expression.
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Breast carcinomas expressing E- and P-cadherin are associated with poor patient survival.
p120-catenin cytoplasmic accumulation is associated with tumours expressing both E- and P-cadherin.
The cytoplasmic localisation of p120-catenin may play an important role in mediating the tumourigenic effects derived from P-cadherin aberrant expression.
Cadherins mediate one of the most important types of adhesive interactions required for the maintenance of normal tissue architecture.1 2 Among the cadherin superfamily, classical cadherins, such as epithelial (E-), neural (N-) and placental (P-) cadherin, are the best characterised adhesion molecules, presenting three distinct domains (extracellular, transmembrane and cytoplasmic) and promoting mainly homophilic and homotypic calcium-dependent interactions.3 Classical cadherins form tight complexes with catenins, a group of cytoplasmic proteins which link the cadherin cytoplasmic tail to the actin cytoskeleton and facilitate clustering into the junctional structure.4 These cytoplasmic partners include β-, γ- and p120-catenins (βctn, γctn and p120ctn), which in turn bind α-catenin (αctn) that interacts directly or indirectly with the actin filament network.5
Cadherin-mediated adhesion is frequently altered during tumour progression, facilitating tumour-cell migration, invasion and metastatic dissemination.6 E-cadherin, the major cell–cell adhesion protein in epithelial tissues, functions as a tumour suppressor and is often down-regulated in human epithelial cancers.7 Particularly in breast carcinomas, the lack or decreased expression of E-cadherin has been correlated with tumour grade, invasiveness and poor prognosis,8 9 In addition, E-cadherin loss characterises lobular breast carcinomas, and it has been attributed to many defects in E-cadherin gene (CDH1), including inactivating mutations, loss of heterozygosity on chromosome 16q and promoter hypermethylation.10 11 However, repression of E-cadherin gene by transcription factors such as SNAIL, SLUG or ZEB, among others, is another well described mechanism12–18 and has been shown to play a role in ductal carcinomas,19 as well as in lobular carcinomas.20 In addition to these repression mechanisms, post-translational inactivation of E-cadherin has also been studied, where induction of metalloproteases and other proteases by cell matrix contact has a role.21 22 In contrast to epithelial cadherin, P-cadherin expression is usually related to tumourigenic properties, enhancing cell invasion and tumour aggressiveness, and indicating a worse prognosis in breast cancer patients.23–26 The only mechanism that has been proposed to regulate the P-cadherin gene is promoter hypomethylation.26 Further research is still required to elucidate the role played by this protein in human mammary tumours.
Changes in junctional catenin expression may also compromise cadherin-mediated adhesion, increasing cell malignant properties such as invasive and metastatic capacity. In fact, altered expression of αctn, βctn and γctn has been reported to be associated with E-cadherin loss or decreased expression, either in breast carcinoma samples or in breast cancer cell lines.27–29 These alterations were strongly correlated with increased tumour size, metastasis and poor prognosis.30–33 Recently, it was also shown that nodal metastasis might re-express E-cadherin, αctn, βctn and γctn; this re-expression of adhesion proteins may be related to the resettling of circulating tumour cells at metastatic sites, by promoting the intercellular adhesion required for metastatic tumour progression.34 Regarding p120ctn, it is well established that its cytoplasmic accumulation together with E-cadherin loss characterises lobular breast carcinomas, and regulates the invasive phenotype of breast cancer cell lines.35 36 In ductal invasive breast carcinomas, reduction of E-cadherin and βctn is associated with reduced expression of p120ctn35; another study suggested that regulation of this catenin is independent from E-cadherin, αctn and βctn.37
In the present study, we have shown that the normal membrane localisation of p120ctn and βctn is frequently altered in invasive breast carcinomas. Interestingly, we found that the majority of E- and P-cadherin expressing tumours are related to cytoplasmic expression of p120ctn and βctn, this group of breast carcinomas being the one with poor patient survival. This result may explain the poor patient survival and higher aggressiveness of ductal breast carcinomas which maintain E-cadherin and aberrantly express P-cadherin.
MATERIALS AND METHODS
A series of 149 formalin-fixed paraffin-embedded blocks of invasive breast carcinomas were retrieved from the histopathology files at the Department of Pathology, Hospital Xeral Cíes, Vigo, Spain. The series had previously been studied with respect to age, tumour size, lymph node metastasis, histological type and grade, oestrogen receptor-α (ER-α) status, and E- and P-cadherin expression26 (see table 1).
Immunohistochemical staining was performed using the streptavidin–biotin–peroxidase technique (Lab Vision Corporation, Fremont, California, USA) on all glass slides. Heat-induced antigen retrieval was performed using a citrate buffer solution, 10 mM, pH 6.0, at 98°C for 30 min. After washes in a phosphate buffer solution (PBS), the slides were incubated in a 3% hydrogen peroxide solution in methanol (Merck, Merck KgaA, Darmstadt, Germany) to block endogenous peroxidase activity. The slides were covered with a blocking serum (Lab Vision) for 10 min and then incubated with the primary antibody for 2 h at room temperature. Anti-p120ctn and anti-βctn monoclonal antibodies (BD Biosciences, San Diego, California, USA) were applied at dilutions of 1:500 and 1:1200, respectively. After washes in PBS, the slides were incubated with a specific kit based on streptavidin–biotin–peroxidase complex (Imuno Kit UltraVision Detection System, Lab Vision) for 15 min. 3,3-Diaminobenzidine tetrahydrochloride (DAB solution, Dako Corporation, Carpinteria, California, USA) was used as a chromogen, incubating each slide for 10 min. Tissues were then counterstained with Mayer’s haematoxylin (Merck), dehydrated and coverslipped using a permanent mounting solution (Entellan Neu, Merck).
Paraffin sections of normal skin and normal mammary tissue were included as positive controls and to ensure consistency between consecutive runs. Immunohistochemical expression was not assessed in some cases (16 for p120ctn and 13 for βctn), since there was no more tumour material available.
Immunoreactivity was scored according to the subcellular localisation of p120ctn and βctn.35 Four categories were defined, depending on the predominant expression pattern: normal membrane (complete membrane staining), reduced membrane (incomplete membrane staining), cytoplasm (diffuse cytoplasm without any appreciable membrane staining) and negative (no staining).
Contingency tables and the χ2 test were used to estimate the relationship between staining patterns of p120ctn and βctn and the several parameters analysed, such as tumour grade and histological type, lymph node metastasis, ER-α content, and P- and E-cadherin expression. Analysis of variance was used to evaluate differences in tumour size, with 95% CI. Since we had follow-up information for 81 patients, which we defined as the time between first diagnosis and patient death by breast cancer, we estimated the univariate survival curves by the Kaplan–Meier method and compared them using the log-rank test. However, we only had follow-up information for 7 patients with tumours lacking βctn expression, 4 expressing p120ctn normal membranous staining and none without p120ctn staining; thus, to avoid bias in the statistical method used, we did not consider them in the survival analysis. Censored data correspond to those patients that left the study before the end of the considered follow-up. Statistical analysis was performed using SPSS V.15.0 for Windows (SPSS, Inc., Chicago, Illinois, USA). Statistical significance was set at p <0.05.
With regard to the subcellular localisation of p120ctn and βctn, we observed that the staining pattern for both catenins was frequently altered in invasive breast carcinomas (fig 1 and table 1). Of the 133 cases stained for p120ctn, the great majority (121 cases) showed cytoplasmic or reduced membranous expression, and only 10 cases retained this protein at the cell membrane. Similarly, βctn exhibited a reduced membranous or cytoplasmic staining in approximately 70% of the 136 stained cases, and maintained a normal membrane pattern of expression in 33 cases. Complete loss of catenin expression was rarely observed.
Statistical analysis was then performed to determine possible associations between catenin localisation and the available clinicopathological parameters (table 2). We found an association between βctn cytoplasmic localisation and increased tumour size (p<0.001); this tendency was also registered for p120ctn, but was not statistically relevant (p = 0.136). Catenin subcellular distribution was also related to tumour histological type (p = 0.017 for p120ctn; p<0.001 for βctn): invasive ductal carcinoma, not otherwise specified (IDC, NOS) and medullary carcinomas frequently exhibited reduced membranous or cytoplasmic staining for both catenins; all invasive lobular carcinomas (ILCs) presented either cytoplasmic expression of both catenins, or lack of βctn expression; tubular and mucinous carcinomas generally displayed normal or membranous staining for both catenins; apocrine carcinomas mainly exhibited catenin cytoplasmic expression; papillary and cribriform carcinomas presented reduced membranous staining for p120ctn, whereas βctn was heterogeneously distributed. Tumour histological grade was not associated with p120ctn subcellular localisation (p = 0.759), but it was significantly related to βctn (p = 0.022), which generally remained at the membrane in grade I carcinomas and accumulated at the cytoplasm in grade III carcinomas. Additionally, no significant correlation was found between axillary lymph node metastasis and p120ctn localisation, but a trend to a significant association was found with βctn (p = 0.052).
We also observed that cytoplasmic localisation of p120ctn and βctn was highly associated with the lack of ER-α expression (p = 0.005 for both; table 2). Concerning cadherin expression, we observed that E-cadherin positive tumours generally maintained catenin membranous staining, while the absence of E-cadherin was significantly related to cytoplasmic localisation of both catenins (p = 0.044 and p<0.001 for p120ctn and βctn, respectively; table 2). In contrast, P-cadherin negative tumours preserved both catenins at the cell membrane, whereas P-cadherin expression was associated with catenin cytoplasmic accumulation, although these differences did not reach statistical significance (p = 0.128 for p120ctn, and p = 0.173 for βctn).
Considering the co-expression of P- and E-cadherin (tables 3 and 4), we observed relevant associations with p120ctn and βctn subcellular distribution (p = 0.002 and p = 0.004, respectively). In fact, βctn was preferentially localised at the membrane in E-cadherin positive tumours, either in the absence or presence of P-cadherin, and accumulated at the cell cytoplasm in tumours lacking E-cadherin expression. Interestingly, P-cadherin negative and E-cadherin positive tumours generally maintained a membranous expression of p120ctn, whereas those expressing P-cadherin exhibited cytoplasmic immunostaining of this catenin, particularly in the presence of E-cadherin.
Comparing only the tumours that maintain the normal expression of E-cadherin, with or without the aberrant P-cadherin expression, we could see that cytoplasm p120ctn is essentially related to tumours that express both cadherins; the tumours just expressing E-cadherin maintain the p120ctn normal membrane staining (p = 0.0032, data not shown). In contrast, when we evaluated these same tumours for βctn staining, the majority maintain a normal catenin expression, showing that E-cadherin maintenance is prevalent and important to the stabilisation of βctn expression at the membrane; the expression of P-cadherin is not enough to alter its membrane localisation (p = 0.3587, data not shown).
We have previously reported that P-cadherin overexpression is significantly associated with shorter patient survival.15 We therefore carried out a statistical analysis to determine the influence of P- and E-cadherin expression and catenin subcellular distribution on patient overall survival. Interestingly, we observed that tumours expressing both cadherins were clearly associated with poor patient survival (p<0.001, fig 2A). Moreover, Kaplan–Meier plots showed that the probability of survival was considerably lower for patients with tumours with cytoplasmic accumulation of p120ctn (p<0.001, fig 2B) and βctn (p = 0.017, fig 2C).
Catenin altered expression has been reported in a variety of tumour types, being associated with loss of E-cadherin immunoreactivity and with an invasive and metastatic phenotype.8 39 In this study, we investigated the expression and subcellular localisation of p120ctn and βctn in a series of human invasive breast carcinomas, showing that both catenins frequently exhibit a reduced membranous or cytoplasmic staining pattern. These alterations were significantly correlated with lack of E-cadherin and ER-α expression. Regarding βctn, it was possible to associate its cytoplasmic expression with higher histological grade, tumour size and nodal status, suggesting a relevant role for this catenin as a prognostic factor. We were not able to find any tumour with nuclear expression for these catenins, although both have been already described as possibly accumulating at the nuclei, cooperating with specific transcription factors and inducing activation of the Wnt pathway. Usually, this nuclear localisation is related to an aggressive tumour phenotype, negatively affecting patients’ overall survival.40–43
Catenin distribution was also highly correlated with tumour histological types. Tubular and mucinous carcinomas generally displayed a normal or membranous staining pattern for both catenins, which is in accordance with several studies that have already pointed to the preservation of catenin and E-cadherin membrane expression in these carcinomas.44 45 In contrast, all ILCs either presented cytoplasmic expression for both catenins, or did not express βctn, which is also consistent with previous reports showing that altered catenin localisation and complete loss of E-cadherin expression or function is one of the most striking characteristics of lobular tumour types.35 44 45 IDC, NOS and medullary carcinomas frequently exhibited reduced membranous or cytoplasmic staining for both catenins. However, the true loss of E-cadherin expression is a less frequent event in these carcinomas, which does not explain completely the altered localisation of catenins that was observed.45–47 Since we had previously described that these tumours can also express P-cadherin, we decided to evaluate whether the aberrant expression of this protein could somehow be implicated in this discrepancy. Nevertheless, we did not find an association between P-cadherin expression and catenin localisation, but conversely to what happens with E-cadherin, there was a tendency for P-cadherin positive tumours to show a cytoplasmic expression of both catenins. Considering these differences, we decided to evaluate E- and P-cadherin co-expression and correlate it with p120ctn and βctn subcellular localisation. Interestingly, E-cadherin positive and P-cadherin negative tumours showed membrane expression of p120ctn and βctn, whereas those tumours negative for both cadherins presented cytoplasmic p120ctn and a reduced membrane staining of βctn. Analysing P-cadherin positive tumours, we found a correlation with cytoplasmic expression of both catenins. This result seems to be even more specific for p120ctn, where this association was maintained independently from the E-cadherin expression. Additionally, when we analysed the survival curves of these patients, we found that E- and P-cadherin positive tumours, as well as those with catenin cytoplasmic expression, present a worse prognosis. Although the number of patients from which we have survival information is quite limited, it is interesting to see that these results collectively indicate that, also in this series, P-cadherin expression has a relevant role in the prognosis of invasive breast carcinomas, but in tumours maintaining E-cadherin expression.
P-cadherin up-regulation has been reported as an indicator of poor patient survival in breast cancer, mainly in IDC-NOS, medullary and metaplastic carcinomas.24 26 This expression was associated with high histological grade and ER-α negative tumours.26 48 Some studies have shown that these poorly differentiated tumours retain E-cadherin expression, being as invasive and metastatic as those that lack this cadherin.37 49 50 Based on this, we hypothesise that E-cadherin is probably important to maintain the aggregation between cancer cells during the invasion and metastatic process, and P-cadherin is the driving force to make the cells invasive, being important to destabilise the strong adhesion between cells and making them more prone to move. Previously, we reported that P-cadherin cDNA transfection of MCF-7/AZ breast cancer cells, which express E-cadherin wild-type, was able to induce cancer cell invasion through collagen type I and Matrigel artificial extracellular matrixes, demonstrating a pro-invasive activity of this cadherin.25 We also showed that this effect was dependent on the presence of its intact juxtamembrane domain, and more specifically from the binding site of p120ctn.25 In the present study, we observed that aberrant P-cadherin expression in E-cadherin positive breast cancer cells is correlated with destabilisation of the normal cadherin/catenin complex, because it is essentially related to p120ctn cytoplasmic expression. p120ctn cytoplasmic accumulation induced by P-cadherin overexpression has already been reported by Taniuchi et al51 in pancreatic ductal adenocarcinoma cells. Once in the cytoplasm, p120ctn can inhibit Rho A and activate two other Rho GTPases, Rac1 and Cdc42, altering the actin cytoskeleton polymerisation and promoting cell migration and motility.52 This probably explains the invasive phenotype that was found in P-cadherin-expressing breast cancer cells.
However, although P-cadherin was correlated with poorer prognosis, transient and reversible inactivation of the E-cadherin–catenins complex by factors secreted by the tumour cells and/or stromal cells, like metalloproteases, cannot be ruled out based only on the positivity for P-cadherin. Such factors can inactivate the cadherin complex; these post-translational mechanisms can also be related to accumulation of catenins in the cytoplasm.21 22 53 54 The analysis of such factors was beyond the scope of the current study; however, it can be also an alternative for the driving of the described tumour aggressiveness in tumours co-expressing P- and E-cadherins, whether as an isolated mechanism or in synergism with P-cadherin/p120ctn.
In conclusion, the cytoplasmic localisation of catenins (essentially p120ctn) may play an important role in mediating the tumourigenic effects derived from P-cadherin aberrant expression, including enhanced motility and invasion, particularly in tumours which preserve E-cadherin expression. Since P-cadherin expression is able to identify breast carcinomas with a basal-like phenotype,55–58 which are tumours with a very aggressive behaviour and with a worse prognosis to the patients, it would be interesting to see how p120ctn and βctn localisations are related with breast cancer genetic profiles, in order to understand whether the Wnt pathway can be responsible for the genesis of these tumours.
Competing interests: None.
Funding: This work was supported by three research grants (JP: SFRH/BPD/15319/2005; ALC: POCI/N/07.01.02/10/25/2005; ASR: POCI/BIA-BCM/59252/2004) and by a scientific project (POCI/BIA-BCM/59252/2004), all financed by the Portuguese Science and Technology Foundation. We are grateful to the Calouste Gulbenkian Foundation for the “Programa Gulbenkian de Estímulo à Investigação (FCG 55/05)”.
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