Expression analysis of E-cadherin, Slug and GSK3β in invasive ductal carcinoma of breast

Breast cancer is a major cause of female mortality in the Western world. In India, it is the second most common cancer among females, while in the metropolitan cities; it ranks as the most common cancer [1]. The incidence rate is low as compared to the west with an age-adjusted incidence of 19.1 per 100,000 women and a crude incidence of 16.5 per 100,000 women [2]. The key biological processes such as embryonic development, tissue remodeling, restitution and wound repair, require epithelial cells to escape from the rigid structural constraints of the tissue architecture and adopt a phenotype more amenable to cell migration and movement. The highly conserved and fundamental process that achieves this morphogenetic transformation is known as epithelial-mesenchymal transition (EMT) [3, 4]. The progression of tumors to invasive cancers and metastatic disease also involves localized occurrence of EMT [5, 6].

Loss of E-cadherin mediated cell adhesion is one of the key mechanisms involved in metastatic conversion of epithelial cells and EMT [7, 8]. Numerous studies have described a partial or complete loss of E-cadherin during cancer progression [9‐12], which is often correlated with an unfavorable prognosis [13, 14], confirming E-cadherin to be a caretaker of the epithelial state. One of the probable mechanisms involved in E-cadherin dysfunction, especially loss of its expression and consequent promotion of tumor progression is through β-catenin signaling. Several other mechanisms of E-cadherin downregulation have been described. Mutations have been found in the CDH1 gene in about 50% of lobular carcinomas of the breast [15], while ductal breast cancers show heterogeneous loss of E-cadherin expression, associated with epigenetic transcriptional downregulation. Analysis of CDH1 methylation in breast cancers and other tumor types has shown that aberrant hypermethylation of CpG islands in CDH1 promoter region often occurs prior to invasion, indicating it to be an early event in tumorigenesis [16]. Besides regulation of CDH1 by promoter methylation and/or genetic alterations, direct transcriptional control of CDH1 has emerged as an important regulatory mechanism of E-cadherin expression. The proteins Snail, Slug, and Twist have been recently characterized as transcriptional repressors of E-cadherin in breast carcinoma and are regulated by Wnt/β-catenin signaling [17, 18]. Slug expression has been shown to correlate more strongly than snail expression with loss of E-cadherin in breast cancer cell lines, suggesting Slug to be a likely in vivo repressor of E-cadherin expression in breast carcinoma [19].

The key components of EMT and Wnt pathways are schematically depicted in Figure 1. Recent results obtained in our laboratory showed that canonical Wnt signaling is one of the signaling pathways possibly involved in the control of the migration/invasive behavior of Invasive ductal carcinoma of breast (IDCs) [20, 21]. We demonstrated the disorganization of E-cadherin-β-catenin complexes and the regulation of vimentin expression by β-catenin mediated pathway in IDCs, thereby supporting the notion that Wnt/β-catenin signaling is implicated in regulation of EMT [21].

E-cadherin, Slug and Glycogen synthase kinase 3β (GSK3β) play important roles in EMT transition via Wnt/β-catenin signaling. GSK3β resides at the junction of PI3K/AKT and Wnt/β-catenin/TCF survival pathways, thereby serving critical roles in cellular metabolism, growth and proliferation [22, 23]. Under un-stimulated conditions, GSK3β pools are constitutively active, but they are phospho-inhibited upon PI3K/AKT or Wnt activation [24], resulting in cytoplasmic and nuclear accumulation of β-catenin.

Recent studies from our laboratory as well as by others substantiate the role of active Wnt/β-catenin pathway in proliferation and induction of EMT in tumor cells [5, 20, 21]. Taking our previous findings into consideration, the primary aim of this study was to determine the expression patterns of integral components of the canonical Wnt/β-catenin signaling- GSK3β, E-cadherin and Slug in IDCs and their associations with clinicopathological parameters for better understanding the biological and clinical relevance of Wnt/β-catenin pathway activation in sporadic breast cancer.

Recent studies demonstrate that canonical Wnt signaling pathway is associated with both stem cell and tumor cell development [27‐29]. 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, 21, 31]. 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, 33]. 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 [34‐36]. 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 [9‐12].

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 [25]. 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, 37]. 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 3). Our result is in the agreement with the previous studies, showing total loss of E-cadherin protein in ILCs [11, 12]. 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.

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, 38, 39]. 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, 40]. 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 2).

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⁺/CD24^- population harbors stem cell properties. These CD44⁺/CD24^- 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, 43, 44]. 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.

In the present study, we provide clinical evidence in support of up-regulation of Wnt/β-catenin signaling in invasive ductal carcinoma of breast and key components of this signaling pathway such as E-cadherin, Slug, GSK3β and β-catenin to be associated with Epithelial Mesenchymal Transition (EMT) process and pathogenesis of IDCs. Moreover, a better understanding of the pathways (such as the Wnt/β-catenin signaling pathway) that trigger EMT and cancer cell self renewal (cancer stem cells) might lead to new therapeutic approaches for cancer patients by developing molecular tools that interfere with them.

The authors declare that they have no competing interests.