E‐cadherin deregulation in breast cancer

Abstract E‐cadherin protein (CDH1 gene) integrity is fundamental to the process of epithelial polarization and differentiation. Deregulation of the E‐cadherin function plays a crucial role in breast cancer metastases, with worse prognosis and shorter overall survival. In this narrative review, we describe the inactivating mechanisms underlying CDH1 gene activity and its possible translation to clinical practice as a prognostic biomarker and as a potential targeted therapy.


| INTRODUC TI ON
The E-cadherin (E-cad) gene (CDH1) [OMIM + 192090] is a calcium-dependent cell-to-cell adhesion molecule and tumour suppressor protein that is the only germline molecular defect associated with hereditary diffuse gastric and lobular breast cancers. 1,2 The E-cad protein plays a critical role in establishing and maintaining polarized and differentiated epithelia through intercellular adhesion complexes. This molecule is considered an invasion suppressor, and its deregulation is often found in advanced cases of some epithelial carcinomas. 3 Deletion or deregulation of E-cad is also correlated with the infiltrative and metastatic ability of the tumour because of disruption of the cadherin-catenin complex, with consequent loss of cell adhesion and concomitant increase in cell motility. 4,5 In gastric carcinoma in particular, defective mechanisms of E-cad are well associated with cancer metastatization; patients carrying any somatic E-cad alterations show the worst prognosis and shortest probability of overall survival (OS). 6 In relation to breast cancer, regular E-cad function presents as an inhibitor of metastasis. It has been shown that somatic E-cad inactivation is associated with an aggressive pattern of breast cancer, particularly lymphovascular invasion and metastasis in the axillary lymph nodes. [7][8][9][10] Cancer metastatization often represents a dramatic event for the patient, due to the psychological impact of its worse prognosis and shorter OS. Prognostic molecular biomarkers are fundamental to assess the risk of tumour relapse after curative intent and cancer metastatization in this setting.
In this narrative review, we describe the principal mechanisms of E-cad inactivation (genetic and epigenetic) and its possible clinical implications for breast cancer as a prognostic and therapeutic factor.

| MECHANIS MS OF E-C AD INAC TIVATI ON
Breast cancer's progression and capacity to invade and metastasize to distant sites is strongly associated with the loss of E-cad. It has been postulated that loss of E-cad expression is an early gatekeeper event in in situ lobular breast cancer and a precursor of invasive lobular breast cancer. 7,11 Over the last several years, a number of mechanisms have been identified as the cause of E-cad inactivation in breast cancer. [12][13][14] Inactivating mutations in the CDH1 gene have been frequently described and can explain a great proportion of invasive lobular breast cancer cases. Indeed, one of the first studies addressing the contribution of CDH1 mutations to the absence of E-cad expression found that 55% (21 of 38 cases) of breast cancers from the lobular subtype harboured CDH1 genetic alterations. 15 In a subsequent study, Berx and colleagues 13 12 6 novel mutations were detected in a set of 22 invasive lobular carcinomas, accounting for 27% of the cases analysed. Interestingly, in the majority of these studies, mutations were found in combination with loss of heterozygosity (LOH) at the E-cad chromosomal locus, spotlighting E-cad as a tumour suppressor gene. 12,13,16 Specifically, among the 23 mutations identified by Berx and colleagues, 13 21 were found in combination with LOH of the wild typeCDH1locus. These data were further validated by Huiping and colleagues, 16 who detected LOH at 16q22.1 in all of the lobular breast tumours examined by their study. Currently, it is well established that 50% of invasive lobular carcinomas show LOH, which is determinant for protein dysfunction and loss of expression. 12,13,17 Apart from structural genetic alterations, epigenetic modifications have also emerged as a possible cause for the impairment of E-cad expression and function. In particular, hypermethylation of the CDH1 promoter is the predominant mechanism of E-cad loss in multiple types of cancer, including breast cancer. [18][19][20] In this context, Shargh and colleagues 21 investigated the association between CDH1 promoter methylation and E-cad expression in 50 ductal breast cancer cases and their respective paired normal breast samples. They observed that 94% of ductal breast cancers were methylated at the CDH1 promoter. Furthermore, they found that there was no detectable E-cad expression in all the cases displaying complete promoter methylation.
Clinically, an association between CDH1 methylation and breast cancer progression has also been reported. Nass et al 22 examined 111 cases of ductal breast carcinomas and observed that CDH1 methylation was present in 31% of in situ lesions, in 52% of invasive tumours, and in 61% of metastatic cancers, indicating an increase of CpG island methylation as cells gain invasiveness and metastatic potential. Later on, the impact of hypermethylation on 6 tumour suppressor genes in tumour progression was evaluated using a series of 151 primary breast tumours, in which the CDH1 promoter was found to be hypermethylated in 53% of cases. Strikingly, in cases with sentinel lymph node metastasis, CDH1 was the most frequently methylated gene (90%), reinforcing the evidence that CDH1 hypermethylation prevails at a more advanced disease stage. 23 Sebova and colleagues 24 proposed that CDH1 hypermethylation can be used as a biomarker for potentially metastasizing tumours. They observed that CDH1 promoter hypermethylation was preferentially observed in breast cancer cases with positive lymph node metastasis and in cases from more aggressive immunohistochemical subtypes.
A correlation between CDH1 methylation status and the prognosis of breast cancer patients was also explored using a series of 137 primary breast cancers, 85 matched normal breast tissue samples, and 13 lung metastasis cases. It was observed that 40.9% of breast cancers and 61.5% of lung metastasis samples were hypermethylated in the CDH1 promoter, while none of the normal breast samples displayed CDH1 methylation. In addition, patients with CDH1 methylation presented significantly poor OS as well as lower disease-free survival (DFS), supporting the significance of CDH1 expression as a predictive biomarker of poor prognosis in breast cancer. 25 At the transcriptional level, several molecules are known to bind to specific DNA sequences of the E-boxes of the CDH1 promoter, repressing the transcription of E-cad and activating mesenchymal genes, and thus promoting the so called 'epithelial to mesenchymal transition' (EMT). 26 This loss of epithelial gene expression and activation of a mesenchymal molecular profile can involve SNAIL, zinc finger E-box-binding (ZEB) and TWIST transcription factors, whose expression is critical to cancer development. 27,28 In breast cancer, it has been reported that silencing of SNAIL increases E-cad expression and, consequently, decreases expression of mesenchymal markers, decreases tumourigenicity and inhibits the invasive behaviour of breast cancer cells. 29 Figure 1 provides an illustration of the mechanisms underlying E-cad inactivation in breast cancer.

| E-C AD A S PROG NOS TI C B I OMARK ER
Breast cancer is a highly heterogeneous disease in which even simi-  The presence of CDH1 epigenetic and structural alterations in a diagnostic/pre-operative biopsy may provide clinically useful information to improve patient management, particularly to infer the prognosis of breast cancer and the pattern of tumour dissemination.

| P OTENTIAL ROLE OF E-C AD A S A THER APEUTI C TARG E T
To date, E-cad is not a molecular target for therapeutic intervention; The heterodimer E-cad-epidermal growth factor receptor (EGFR) complex is another attractive therapeutic target. The presence of extracellular E-cad mutations disturbs the stability of E-cad-EGFR heterodimers, allowing for receptor activation by the ligand and consequent activation of the RhoA signalling pathway, accompanied by enhanced cell motility. Upon interaction with EGFR, E-cad exerts an inhibitory function that modulates the kinase activity of the receptor in an adhesion-independent manner. The extracellular E-cad mutants, by reducing its affinity for EGFR, increase the fraction of unbound EGFR, which can thus be activated, resulting in enhanced cell motility. This effect is transmitted through the activation of RhoA. 48 Further study is needed to clarify the role of HER2 in this pathway and the effect of targeted treatment using HER2 inhibitors (ie trastuzumab).

F I G U R E 1
Mechanisms underlying E-cad inactivation in breast cancer. Loss of E-cad expression and the spreading abilities of breast cancer cells have been associated with mutations in CDH1 gene, loss of heterozygosity at the E-cad chromosomal locus, hypermethylation of the CDH1 promoter, transcriptional repression and post-translational modifications, such as aberrant glycosylation

| HOW TO MANAG E E-C AD IN THE CLINIC AL S E T TING
E-cad may play a potential strategic role in the clinical management of breast cancer patients as a predictor of prognosis and survival. In this review, we analyzed several studies reporting that E-cad dysfunction is associated with worse prognosis and shorter OS. Although mechanisms of CDH1 inactivation are still complex to manage routinely, it is reasonable to evaluate E-cad expression in breast tumours using standard methods, such as immunohistochemistry. We have already demonstrated that CDH1 inactivation is a suitable predictive biomarkers of prognosis in gastric cancer's patients. In particular, the worst patient survival rate among all cases analysed was seen in patients with tumours carrying CDH1 structural alterations. 6 In normal breast tissue, epithelial cells show strong and complete membranous expression of E-cad. 49 Among breast cancers, nearly 90% of invasive lobular carcinomas display complete or partial loss of E-cad immunohistochemical expression that is considered an important (but not necessary) diagnostic feature for this histological subtype (Figure 2). 50 (Figure 3).

| CON CLUS ION
In this narrative article, we have provided evidence for the aggres-

ACK N OWLED G EM ENTS
This manuscript was supported by a grant from the Italy Ministry of Health (Project Code GR-2016-02361655), in part by the Ricerca Corrente and 5x1000 funds, and in part by the Fondazione IEO-CCM.

CO N FLI C T O F I NTE R E S T
The authors have no potential conflicts of interest to declare.

AUTH O R CO NTR I B UTI O N S
GC, SPD, FC, AG, AI, EGR and SG researched data for this article. and EGR wrote the manuscript. All authors reviewed and/or edited the manuscript before submission.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.