Geert Berx

The Two-Handed E Box Binding Zinc Finger Protein SIP1 Downregulates E-Cadherin and Induces Invasion

Transcriptional downregulation of E-cadherin appears to be an important event in the progression of various epithelial tumors. SIP1 (ZEB-2) is a Smad-interacting, multi-zinc finger protein that shows specific DNA binding activity. Here, we report that expression of wild-type but not of mutated SIP1 downregulates mammalian E-cadherin transcription via binding to both conserved E2 boxes of the minimal E-cadherin promoter. SIP1 and Snail bind to partly overlapping promoter sequences and showed similar silencing effects. SIP1 can be induced by TGF-beta treatment and shows high expression in several E-cadherin-negative human carcinoma cell lines. Conditional expression of SIP1 in E-cadherin-positive MDCK cells abrogates E-cadherin-mediated intercellular adhesion and simultaneously induces invasion. SIP1 therefore appears to be a promoter of invasion in malignant epithelial tumors.

Regulatory networks defining EMT during cancer initiation and progression

Epithelial to mesenchymal transition (EMT) is essential for driving plasticity during development, but is an unintentional behaviour of cells during cancer progression. The EMT-associated reprogramming of cells not only suggests that fundamental changes may occur to several regulatory networks but also that an intimate interplay exists between them. Disturbance of a controlled epithelial balance is triggered by altering several layers of regulation, including the transcriptional and translational machinery, expression of non-coding RNAs, alternative splicing and protein stability.

E-cadherin is a tumour/invasion suppressor gene mutated in human lobular breast cancers.

Compelling experimental evidence exists for a potent invasion suppressor role of the cell‐cell adhesion molecule E‐cadherin. In addition, a tumour suppressor effect has been suggested for E‐cadherin. In human cancers, partial or complete loss of E‐cadherin expression correlates with malignancy. To investigate the molecular basis for this altered expression we developed a comprehensive PCR/SSCP mutation screen for the human E‐cadherin gene. For 49 breast cancer patients the occurrence of tumour‐specific mutations in the E‐cadherin gene was examined. No relevant DNA changes were encountered in any of 42 infiltrative ductal or medullary breast carcinoma samples. In contrast, four out of seven infiltrative lobular breast carcinomas harboured protein truncation mutations (three nonsense and one frameshift) in the extracellular part of the E‐cadherin protein. Each of the four lobular carcinomas with E‐cadherin mutations showed tumour‐specific loss of heterozygosity of chromosomal region 16q22.1 containing the E‐cadherin locus. In compliance with this, no E‐cadherin expression was detectable by immunohistochemistry in these four tumours. These findings offer a molecular explanation for the typical scattered tumour cell growth in infiltrative lobular breast cancer.

The cell-cell adhesion molecule E-cadherin

Abstract.This review is dedicated to E-cadherin, a calcium-dependent cell-cell adhesion molecule with pivotal roles in epithelial cell behavior, tissue formation, and suppression of cancer. As founder member of the cadherin superfamily, it has been extensively investigated. We summarize the structure and regulation of the E-cadherin gene and transcript. Models for E-cadherin-catenin complexes and cell junctions are presented. The structure of the E-cadherin protein is discussed in view of the diverse functions of this remarkable protein. Homophilic and heterophilic adhesion are compared, including the role of E-cadherin as a receptor for pathogens. The complex post-translational processing of E-cadherin is reviewed, as well as the many signaling activities. The role of E-cadherin in embryonic development and morphogenesis is discussed for several animal models. Finally, we review the multiple mechanisms that disrupt E-cadherin function in cancer: inactivating somatic and germline mutations, epigenetic silencing by DNA methylation and epithelial to mesenchymal transition-inducing transcription factors, and dysregulated protein processing.

DeltaEF1 is a transcriptional repressor of E-cadherin and regulates epithelial plasticity in breast cancer cells

Downregulation of E-cadherin is a crucial event for epithelial to mesenchymal transition (EMT) in embryonic development and cancer progression. Using the EpFosER mammary tumour model we show that during EMT, upregulation of the transcriptional regulator deltaEF1 coincided with transcriptional repression of E-cadherin. Ectopic expression of deltaEF1 in epithelial cells was sufficient to downregulate E-cadherin and to induce EMT. Analysis of E-cadherin promoter activity and chromatin immunoprecipitation identified deltaEF1 as direct transcriptional repressor of E-cadherin. In human cancer cells, transcript levels of deltaEF1 correlated directly with the extent of E-cadherin repression and loss of the epithelial phenotype. The protein was enriched in nuclei of human cancer cells and physically associated with the E-cadherin promoter. RNA interference-mediated downregulation of deltaEF1 in cancer cells was sufficient to derepress E-cadherin expression and restore cell to cell adhesion, suggesting that deltaEF1 is a key player in late stage carcinogenesis.

Mutations of the human E-cadherin (CDH1) gene

The cell–cell adhesion molecule E‐cadherin is well known to act as a strong invasion suppressor in experimental tumor cell systems. Frequent inactivating mutations have been identified for the E‐cadherin gene (CDH1) in diffuse gastric cancers and lobular breast cancers. To date, 69 somatic mutations have been reported comprising, in addition to few missense mutations, mainly splice site mutations and truncation mutations caused by insertions, deletions, and nonsense mutations. Interestingly, there is a major difference in mutation type between diffuse gastric and infiltrative lobular breast cancers. In diffuse gastric tumors, the predominant defects are exon skippings, which cause in‐frame deletions. By contrast, most mutations found in infiltrating lobular breast cancers are out‐of‐frame mutations, which are predicted to yield secreted truncated E‐cadherin fragments. In most cases, these mutations do occur in combination with loss of heterozygosity (LOH) of the wild‐type allele. Inactivating germline mutations of E‐cadherin were recently reported for families with early‐onset diffuse gastric cancer. Also, at the early stages of sporadic lobular breast and diffuse gastric cancers, E‐cadherin mutations were detected, suggesting loss of growth control by such mutations and defining E‐cadherin as a true tumor suppressor for these particular tumor types. Hum Mutat 12:226–237, 1998.

SIP1/ZEB2 induces EMT by repressing genes of different epithelial cell-cell junctions

SIP1/ZEB2 is a member of the δEF-1 family of two-handed zinc finger nuclear factors. The expression of these transcription factors is associated with epithelial mesenchymal transitions (EMT) during development. SIP1 is also expressed in some breast cancer cell lines and was detected in intestinal gastric carcinomas, where its expression is inversely correlated with that of E-cadherin. Here, we show that expression of SIP1 in human epithelial cells results in a clear morphological change from an epithelial to a mesenchymal phenotype. Induction of this epithelial dedifferentiation was accompanied by repression of several cell junctional proteins, with concomitant repression of their mRNA levels. Besides E-cadherin, other genes coding for crucial proteins of tight junctions, desmosomes and gap junctions were found to be transcriptionally regulated by the transcriptional repressor SIP1. Moreover, study of the promoter regions of selected genes by luciferase reporter assays and chromatin immunoprecipitation shows that repression is directly mediated by SIP1. These data indicate that, during epithelial dedifferentiation, SIP1 represses in a coordinated manner the transcription of genes coding for junctional proteins contributing to the dedifferentiated state; this repression occurs by a general mechanism mediated by Smad Interacting Protein 1 (SIP1)-binding sites.

The E-cadherin/catenin complex: an important gatekeeper in breast cancer tumorigenesis and malignant progression

E-cadherin is a cell–cell adhesion protein fulfilling a prominent role in epithelial differentiation. Data from model systems suggest that E-cadherin is a potent invasion/tumor suppressor of breast cancer. Consistent with this role in breast cancer progression, partial or complete loss of E-cadherin expression has been found to correlate with poor prognosis in breast cancer patients. The E-cadherin gene (CDH1) is located on human chromosome 16q22.1, a region frequently affected with loss of heterozygosity in sporadic breast cancer. Invasive lobular breast carcinomas, which are typically completely E-cadherin-negative, often show inactivating mutations in combination with loss of heterozygosity of the wild-type CDH1 allele. Mutations were found at early noninvasive stages, thus associating E-cadherin mutations with loss of cell growth control and defining CDH1 as the tumor suppressor for the lobular breast cancer subtype. Ductal breast cancers in general show heterogeneous loss of E-cadherin expression, associated with epigenetic transcriptional downregulation. It is proposed that the microenvironment at the invasive front is transiently downregulating E-cadherin transcription. This can be associated with induction of nonepithelial cadherins.

E-cadherin inactivation in lobular carcinoma in situ of the breast: an early event in tumorigenesis

In breast cancer, inactivating point mutations in the E-cadherin gene are frequently found in invasive lobular carcinoma (ILC) but never in invasive ductal carcinoma (IDC). Lobular carcinoma in situ (LCIS) adjacent to ILC has previously been shown to lack E-cadherin expression, but whether LCIS without adjacent invasive carcinoma also lacks E-cadherin expression and whether the gene mutations present in ILC are already present in LCIS is not known. We report here that E-cadherin expression is absent in six cases of LCIS and present in 150 cases of ductal carcinoma in situ (DCIS), both without an adjacent invasive component. Furthermore, using mutation analysis, we could demonstrate the presence of the same truncating mutations and loss of heterozygosity (LOH) of the wild-type E-cadherin in the LCIS component and in the adjacent ILC. Our results indicate that E-cadherin is a very early target gene in lobular breast carcinogenesis and plays a tumour-suppressive role, additional to the previously suggested invasion-suppressive role.

Simultaneous loss of E-cadherin and catenins in invasive lobular breast cancer and lobular carcinoma in situ

Loss of expression of the intercellular adhesion molecule E‐cadherin frequently occurs in invasive lobular breast carcinomas as a result of mutational inactivation. Expression patterns of E‐cadherin and the molecules comprising the cytoplasmic complex of adherens junctions, α‐, β‐ and γ‐catenin, were studied in a series of 38 lobular breast carcinomas with known E‐cadherin mutation status. The effect of loss of E‐cadherin by mutational inactivation (or other mechanisms) on the expression of catenins was investigated. Complete loss of plasma membrane‐associated E‐cadherin expression was observed in 32 out of 38 invasive lobular carcinomas, for which in 21 cases a mutation was found in the extracellular domain of E‐cadherin. In total, 15 frameshift mutations of small deletions or insertions, ranging from 1 to 41 bp, three non‐sense mutations, and three splice mutations were identified. Mutations were scattered over the whole coding region and no hot spots could be detected. In all cases, simultaneous loss of E‐cadherin and α‐ and β‐catenin expression was found; in 50 per cent of these cases, additional loss of γ‐catenin was observed. In six invasive lobular carcinomas, expression of both E‐cadherin and catenins was retained. In none of these carcinomas was an E‐cadherin mutation detected. Lobular carcinoma in situ adjacent to invasive lobular carcinoma showed simultaneous loss of E‐cadherin and catenins in all the cases studied—remarkably, also, in four cases positive for E‐cadherin and catenin expression in the invasive component. These results indicate that simultaneous loss of E‐cadherin and α‐, β‐ and γ‐catenin may be an important step in the formation of lobular carcinoma in situ, as a precursor of invasive lobular breast cancer. Events additional to E‐cadherin inactivation must be involved in the transition of lobular carcinoma in situ to invasive lobular carcinoma.