Friedel Nollet

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.

Predominance of beta-catenin mutations and beta-catenin dysregulation in sporadic aggressive fibromatosis (desmoid tumor).

Aggressive fibromatosis (also called desmoid tumor) occurs as a sporadic lesion or as part of Familial Adenomatous Polyposis, which is caused by germ line mutations in the Adenomatous polyposis Coli (APC) gene. APC is involved in the regulation of the cellular level of beta-catenin, which is a mediator in Wnt signaling. Mutational analysis of the beta-catenin and APC genes was performed in 42 sporadic aggressive fibromatoses. Nine tumors had mutations in APC, and 22 had a point mutation in beta-catenin at either codon 45 or codon 41 (producing a stabilized beta-catenin protein product). Immunohistochemistry showed an elevated beta-catenin protein level in all tumors, regardless of mutational status. Beta-catenin localized to the nucleus, and was not tyrosine phosphorylated in the six tumors in which this was tested. The demonstration of mutations in two mediators in the Wnt-APC-beta-catenin pathway implicates beta-catenin stabilization as the key factor in the pathogenesis of aggressive fibromatosis. This is the first demonstration of somatic beta-catenin mutations in a locally invasive, but non metastatic lesion composed of spindle cells, illustrating the importance of beta-catenin stabilization in a variety of cell types and neoplastic processes. Moreover, this tumor has one of the highest reported frequencies of beta-catenin mutations of any tumor type.

Dysregulation of the E-Cadherin/Catenin Complex by Irreversible Mutations in Human Carcinomas

The different proteins of the E-cadherin/catenin cell-cell adhesion complex are believed to play a predominant role in carcinogenesis. Aberrant expression of these proteins has been found in many different human carcinomas, indicating abnormal regulation. In general, inactivating mutations of the human E-cadherin gene are rare; they are, however, highly frequent in infiltrating lobular breast carcinomas and in diffuse gastric carcinomas. These mutations mostly occur in combination with loss of heterozygosity (LOH) of the wild-type allele. Mutations were found at very early non-invasive stages, thus associating E-cadherin mutations with loss of growth control and defining E-cadherin as a real tumour suppressor for these particular tumour types. Defects affecting both alleles of the alpha E-catenin gene have been found in different human carcinoma cell lines, resulting in the loss of E-cadherin-mediated cell-cell adhesion. Mutations of the beta-catenin gene in colon tumours and melanomas were found to result in an accumulation of the protein in the cytosol. Upon translocation to the nucleus, this beta-catenin enhances TCF/LEF-dependent transcriptional activity. This suggests that mutated beta-catenin can act as an oncogene in these particular tumour types. The multiple interaction partners of beta-catenin are known to be involved in signal transduction, actin organization, protein phosphorylation or transcriptional regulation. This makes this protein an intriguing alternative target for either activation or inactivation in human cancer types characterized by frequent E-cadherin or APC deficiencies.

The αE-catenin gene ( CTNNA1 ) acts as an invasion-suppressor gene in human colon cancer cells

The acquisition of invasiveness is a crucial step in the malignant progression of cancer. In cancers of the colon and of other organs the E-cadherin/catenin complex, which is implicated in homotypic cell-cell adhesion as well as in signal transduction, serves as a powerful inhibitor of invasion. We show here that one allele of the αE-catenin (CTNNA1) gene is mutated in the human colon cancer cell family HCT-8, which is identical to HCT-15, DLD-1 and HRT-18. Genetic instability, due to mutations in the HMSH6 (also called GTBP) mismatch repair gene, results in the spontaneous occurrence of invasive variants, all carrying either a mutation or exon skipping in the second αE-catenin allele. The αE-catenin gene is therefore, an invasion-suppressor gene in accordance with the two-hit model of Knudsen for tumour-suppressor genes.

Did the Four Human Cancer Cell Lines DLD-1, HCT-15, HCT-8, and HRT-18 Originate from One and the Same Patient?

Four human colon cancer cell lines, HCT-8, HRT-18, DLD-1, and HCT-15, with an epithelioid morphotype reproducibly formed alpha-catenin-deficient round cells. Using DNA fingerprinting, we found that these four cell lines have an identical genetic background. Our finding strongly suggests a genetic background for the reproducible loss of alpha-catenin and the ensuing acquisition of invasiveness in all four cell lines.

Expression of the E‐cadherin–catenin complex in lung neuroendocrine tumours

Neuroendocrine tumours (NETs) of the lung represent a wide spectrum of phenotypically distinct entities, with differences in tumour progression and aggressiveness. The redistribution and/or the loss of various cell adhesion molecules, such as the E‐cadherin–catenin complex, play a predominant role in carcinogenesis and in tumour invasion. Moreover, mutations in exon 3 of the β‐catenin gene, the adenomatous polyposis coli (APC) gene or the E‐cadherin genes were previously found to result in intracytoplasmic and/or nuclear β‐catenin protein accumulation, activating nuclear transcription of target genes involved in tumour progression. In the present study, the distribution of the components of this E‐cadherin–catenin complex has been investigated by immunohistochemistry and an attempt has been made to correlate the abnormal expression pattern with the eventual detection of mutations in the corresponding genes. This study included 27 primary NETs of the lung, with nine typical carcinoids (TCs), three atypical carcinoids (ACs), and 15 large cell neuroendocrine carcinomas (LCNECs). The E‐cadherin–catenin complex remained expressed in most of these lung tumours, but with a cytoplasmic and/or nuclear redistribution of β‐catenin, E‐cadherin, and α‐catenin; abnormal positive immunoreactivity was observed in 24 (88.9%), in 21 (80.8%), and in 20 (76.9%) NETs, respectively. In the great majority of cases, there was a good correlation between the expression of these three proteins, but no significant association with histological classification or TNM stage. Thus, E‐cadherin–complex redistribution cannot be considered a prognostic marker in NET of the lung. Of particular interest was the frequent focal β‐catenin nuclear immunostaining (55.5% in total), which was also unrelated to histological type or TNM stage. However, this study failed to detect any mutation in exon 3 of the β‐catenin gene, in the APC gene or in the E‐cadherin gene. These data suggest another mechanism of regulation of β‐catenin in these tumours. Copyright

Genetic alterations involving exon 3 of the β‐catenin gene do not play a role in adenocarcinomas of the esophagus

β‐catenin has been identified as an oncogene. Phosphorylation of sites encoded by exon 3 of the β‐catenin gene facilitates degradation of this protein by the adenomatous polyposis coli (apc) gene product. Mutations in these sites or inactivation of apc lead to stabilization of β‐catenin, which then translocates to the nucleus where it modulates the transcription of genes involved in tumor formation. To explore the role of β‐catenin mutations in adenocarcinomas of the esophagus, we screened for genetic alterations in exon 3 in 69 tumor samples. We detected no mutations in exon 3 by PCR‐SSCP analysis nor did we find large interstitial deletions involving exon 3. β‐catenin immunostaining on 54 tumors showed focal nuclear staining in 7 tumors and homogeneous nuclear staining in 3 tumors; in the latter; no mutations in the mutation cluster region of apc were detected. These results show that genetic alterations of exon 3 of the β‐catenin gene do not occur and therefore do not contribute to the pathogenesis of esophageal adenocarcinomas. The abnormal cytoplasmic and nuclear localization of β‐catenin indicates that other mechanisms leading to elevated free β‐catenin in these cancers must be involved. Int. J. Cancer 86:533–537, 2000.

A Real-Time Polymerase Chain Reaction Assay for Rapid, Sensitive, and Specific Quantification of the JAK2V617F Mutation Using a Locked Nucleic Acid-Modified Oligonucleotide

The JAK2V617F mutation has emerged as an essential molecular determinant of myeloproliferative neoplasms (MPNs). The aim of this study was to evaluate the analytical and clinical performances of a real-time PCR (qPCR) assay using a combination of hydrolysis probes and a wild-type blocking oligonucleotide, all containing locked nucleic acid (LNA) bases. Moreover, we validated a procedure for precise quantification of the JAK2V617F allele burden. We used DNA samples from patients suspected to suffer from MPN and dilutions of HEL cells, carrying the mutation, to compare the LNA-qPCR assay to two previously published methods. All assays detected the same 36 JAK2V617F positive patients of 116 suspected MPN diagnostic samples. No amplification of normal donor DNA was observed in the LNA-qPCR, and the assay was able to detect and reproducibly quantify as few as 0.4% of the JAK2V617F allele in wild-type alleles. Quantification of the JAK2V617F allele burden showed similar proportion levels among the different MPN entities as described by other groups. In conclusion, the LNA-qPCR is a rapid, robust, sensitive, and highly specific assay for quantitative JAK2V617F determination that can be easily implemented in clinical molecular diagnostic laboratories. Moreover, precise quantification allows determination of JAK2V617F burden at diagnosis as well as the evaluation of response to JAK2 inhibitors.

An efficient and reliable multiplex PCR-SSCP mutation analysis test applied to the human E-cadherin gene.

The invasion suppressor gene E‐CADHERIN (CDH1) is downregulated in a large variety of human carcinomas. Up to now, mutational analysis of the CDH1 gene has been described for 325 tumors derived from only four different tissue types. A simple but sensitive mutation detection assay is needed to screen many more tumor types, possibility bearing E‐cadherin inactivating mutations. For that purpose, we developed a multiplex PCR‐SSCP analysis for all 16 CDH1 exons. Ease of experimentation was combined with reliable sensitivity. Indeed, the present multiplex analysis reduces the number of manipulations to 50%, while the mutation detection turned out to be highly efficient and sensitive. Hum Mutat 9:567–574, 1997.

The human αE-catenin gene CTNNA1: mutational analysis and rare occurrence of a truncated splice variant

Abnormal expression of the alphaE-catenin protein, a component of the E-cadherin/catenin cell adhesion complex, is frequently observed in human cancer cells. An inverse correlation between alphaE-catenin expression and tumor malignancy can be of prognostic value. Mutations of the alphaE-catenin gene, CTNNA1, were described in several human cancer cell lines and were found to result in aberrant cell adhesion. We have developed a polymerase chain reaction/single-strand conformation polymorphism-based method for mutation analysis of this gene in human tumor DNA. This approach enabled us to identify several polymorphisms in a set of desmoid tumors, demonstrating that this method is suitable for alphaE-catenin mutational analysis. On the basis of our genomic characterization data, we found that the previously reported alternative splicing of the alphaE-catenin gene actually generates a frame-shift, resulting in a truncated alphaE-catenin protein. This finding is unlike the other alpha-catenin family members alphaN-catenin and vinculin, which show in-frame alternative inserts. Furthermore, real-time quantitative reverse transcriptase-PCR analysis did not reveal relevant expression levels of this alternatively spliced alphaE-catenin variant neither in any human tissue or cell line tested, nor at any mouse developmental stage tested. Thus, contrary to previous notions, alternative splicing with in-frame insertion nearby the C-terminal end of the protein is not a general feature for all members of the alpha-catenin/vinculin family.