Recent studies demonstrate that canonical Wnt signaling pathway is associated with both stem cell and tumor cell development [27
]. In many tissues, where stem cell attributes are under the control of Wnt pathway, aberrant activation of this pathway results in tumor formation [30
]. Our current findings support the accumulating evidence that hyperactive Wnt signaling is associated with development and progression of human breast cancer [20
]. Herein, we provide clinical evidence to demonstrate that alterations in expression of the key components of Wnt/β-catenin pathway- E-cadherin, GSK3β and Slug occur in the pathogenesis of IDCs.
During EMT, the epithelial cells acquire fibroblast-like properties and show reduced intercellular adhesion and increased motility; this process is associated with functional loss of E-cadherin [32
]. Further, down regulation of E-cadherin is the key step towards invasive phase of cancer progression; promoter methylation/transcriptional repression are mechanisms largely responsible for loss of E-cadherin expression in IDCs [34
]. Recent data from our laboratory suggests that absence of E-cadherin is partly attributed to promoter methylation of CDH1
in IDCs; CDH1
hypermethylation frequency was found to be 50% in IDCs [21
]. Taking into account the two major histological subtypes of breast cancer, however, different modes of E-cadherin expression modulation have been found. While infiltrating ductal breast cancers mostly show no or only heterogeneously reduced E-cadherin expression, infiltrative lobular breast carcinomas (ILC) are, in most cases (95%), completely E-cadherin-negative [9
In the present study, we observed loss/or reduced expression of E-cadherin in 54% (53/98) of IDCs. Similar E-cadherin loss was reported in 85% cases in a series of 71 ductal carcinomas and correlated with promoter methylation of CDH1
]. In our previous study in the same patient cohort, we analyzed the expression patterns of β-catenin, Disheveled and CyclinD1. Of the 98 IDCs analyzed, loss of cell surface β-catenin was observed in 66% cases, whereas nuclear expression was observed in 44% tumors [20
]. In the current study, we found significant positive association between membranous E-cadherin and β-catenin loss (p = 0.0001) in IDCs. On this basis, we postulate that E-cadherin loss promotes tumorigenesis by effectively releasing membrane bound β-catenin into the cytosol, there by stimulating the canonical Wnt signaling.
Slug mediated loss of E-cadherin expression in IDCs is another finding of our study. Although several transcription factors including Snail and Slug have been implicated in E-cadherin repression, herein we have analyzed the expression of Slug only, because it has been proposed to be a likely in vivo
repressor of E-cadherin as compared to snail in breast carcinomas [19
]. Nuclear accumulation of Slug was observed in 33 of 98 (34%) IDCs, though lower than reported in a previous study [17
], and correlated inversely with tumor grade (p = 0.01) and loss of membranous E-cadherin expression (p = 0.033) in IDCs. The association between E-cadherin membrane loss and its cytoplasmic accumulation with nuclear Slug prompts us to speculate that Slug may act as a transcriptional suppressor of E-cadherin and regulate its cellular turnover in IDCs. Furthermore, significant associations between nuclear and cytoplasmic β-catenin and Slug (p = 0.001; p = 0.005), underscores the importance of β-catenin mediated regulation of Slug in invasive ductal carcinoma of breast. In addition, we also performed Immunohistochemistry for E-cadherin and Slug proteins on 5 histologically confirmed cases of Invasive lobular carcinomas (Figure ). Our result is in the agreement with the previous studies, showing total loss of E-cadherin protein in ILCs [11
]. However, we did find nuclear accumulation of slug in 3 of 5 ILCs. Till date there is no informative study on Slug protein in ILCs, therefore future studies are warranted in this specific area for defining/or elucidating the role of Slug in ILCs.
Figure 3 Expression pattern of E-cadherin, Slug and GSK3β proteins in invasive lobular carcinomas of breast. (A& B) Invasive lobular carcinomas showing loss of E-cadherin staining (C) Invasive lobular carcinoma showing nuclear staining for Slug (more ...)
The other important component of Wnt pathway investigated in our study is GSK3β, a multikinase involved in Wnt, Akt and Hedgehog pathways, all of which are involved in determination of cell fate and morphology; inhibition of GSK3β activity or expression results in bonafide EMT [24
]. We observed loss of GSK3β protein in 53% of IDCs, suggesting endogenous suppression of the GSK3β, either due to Wnt or PI3K-kinase, which are frequently activated in IDCs [20
]. In the canonical Wnt/β-catenin pathway, GSK-3β activity in the destruction complex is inhibited through a yet unclear process, leading to the accumulation of β-catenin that translocates to the nucleus and activates transcription by TCF/LEF transcription factors. In our study, a subset of IDCs (35%) showed nuclear accumulation of GSK3β protein. Other plausible reason for nuclear GSK3β accumulation may be an additional regulation inside the cell and complete inhibition of GSK3β may require activation of multiple signaling pathways simultaneously. Further studies on the expression profiles of pGSK3β and pAkt, using phospho-specific antibodies will certainly help in elucidating the role of GSK3β regulation in Invasive ductal carcinomas of breast. However, these mechanisms need to be proven in future studies. We also observed an inverse association of nuclear GSK3β with tumor grade (p = 0.02), suggesting that the initial tumor development probably requires a rapid and effective repression of GSK3β and stabilization of Slug, thereby inhibiting the expression of E-cadherin. Interestingly, nuclear GSK3β showed positive association with ERα expression (p = 0.019), suggesting that GSK3β may regulate the estrogen receptor mediated transcription in subsets of IDCs.
In a recent study, we demonstrated the relationship of loss of E-cadherin and APC proteins with activation of Wnt/β-catenin signaling driving EMT [21
]. We demonstrated therein, that apart from β-catenin, Disheveled also regulates the expression of Vimentin, establishing direct association of Wnt/β-catenin signaling with EMT. Continuing our focus on Wnt/β-catenin signaling and EMT, in the present study we found various relationships among EMT regulators like β-catenin, E-cadherin and Slug in IDCs (Table ).
Our current findings also support the concept that generation of cancer stem cells (CSCs) - the acquisition of the stemness and tumorigenic characters is driven by induction of EMT [41
]. In breast cancer, CD44+
population harbors stem cell properties. These CD44+
cells, express low or undetectable levels of epithelial markers (E-cadherin and β-catenin) and high levels of mesenchymal markers (vimentin and fibronectin), suggesting that these cells have undergone EMT [42
]. Expression of EMT-inducing factors, such as E-cadherin, β-catenin and Slug, has been shown to be associated with breast tumor recurrence and metastasis [37
]. Importantly, Wnt signaling has been recently established to serve as a molecular link between self renewal, EMT, and metastasis in basal-like breast cancers supporting our clinical findings [45
]. In the presence of Wnt signals, β-catenin has been proposed to partner with TCF/LEF to activate target genes, such as Slug and Twist which promote an EMT, repress differentiation, increase tumor seeding and metastasis. Thus Wnt signaling effects Slug and Twist thereby regulating cell-cell adhesion and EMT; it can also connect EMT with cell fate and differentiation. Taken together, we speculate that E-cadherin, Slug and GSK3β could be exploited as markers and pharmacologic or antibody-based therapies targeting the Wnt pathway components, which may not only improve the management of breast cancer, but also affect tumor recurrence and/or metastasis.