E-cadherin mutations and cell motility: A genotype–phenotype correlation

Mateus, Ana; Simões‐Correia, Joana; Figueiredo, Joana; Heindl, Stefan; Alves, Catarina Castro; Suriano, Gianpaolo; Luber, Birgit; Seruca, Raquel

doi:10.1016/j.yexcr.2009.02.020

Cited by 65 publications

(54 citation statements)

References 48 publications

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“…In fact, we have previously shown a genotype-phenotype correlation between the localization of CDH1 missense mutations and their in vitro functional behavior. 50 Cells expressing E-cadherin mutations located at the extracellular domain are more motile, conferring a more aggressive in vitro phenotype, than those affecting the E-cadherin intracellular portion. 36,50 For that reason, E-cadherin mutations localized at the intracellular domain pose additional problems in clinical terms and make it urgent to improve the accuracy of the in vitro methods.…”

Section: Cdh1 Missense Mutations Affect E-cadherin Subcellular Localimentioning

confidence: 99%

“…50 Cells expressing E-cadherin mutations located at the extracellular domain are more motile, conferring a more aggressive in vitro phenotype, than those affecting the E-cadherin intracellular portion. 36,50 For that reason, E-cadherin mutations localized at the intracellular domain pose additional problems in clinical terms and make it urgent to improve the accuracy of the in vitro methods. Being aware of this, the structural E-cadherin model was recently updated and now covers the prodomain, the extracellular domain and the catenin binding domain.…”

Section: Cdh1 Missense Mutations Affect E-cadherin Subcellular Localimentioning

confidence: 99%

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The importance of E-cadherin binding partners to evaluate the pathogenicity of E-cadherin missense mutations associated to HDGC

et al. 2012

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In hereditary diffuse gastric cancer (HDGC), CDH1 germline gene alterations are causative events in 30% of the cases. In 20% of HDGC families, CDH1 germline mutations are of the missense type and the mutation carriers constitute a problem in terms of genetic counseling and surveillance. To access the pathogenic relevance of missense mutations, we have previously developed an in vitro method to functionally characterize them. Pathogenic E-cadherin missense mutants fail to aggregate and become more invasive, in comparison with cells expressing the wild-type (WT) protein. Herein, our aim was to develop a complementary method to unravel the pathogenic significance of E-cadherin missense mutations. We used cells stably expressing WT E-cadherin and seven HDGC-associated mutations (five intracellular and two extracellular) and studied by proximity ligation assays (PLA) how these mutants bind to fundamental regulators of E-cadherin function and trafficking. We focused our attention on the interaction with: p120, b-catenin, PIPKIc and Hakai. We showed that cytoplasmic E-cadherin mutations affect the interaction of one or more binding partners, compromising the E-cadherin stability at the plasma membrane and likely affecting the adhesion complex competence. In the present work, we demonstrated that the study of the interplay between E-cadherin and its binding partners, using PLA, is an easy, rapid, quantitative and highly reproducible technique that can be applied in routine labs to verify the pathogenicity of E-cadherin missense mutants for HDGC diagnosis, especially those located in the intracellular domain of the protein. Keywords: HDGC; E-cadherin; CDH1 mutations; E-cadherin trafficking; E-cadherin binding partners; diagnostic method INTRODUCTIONHereditary diffuse gastric cancer (HDGC) is an autosomal dominant cancer syndrome characterized by a high risk of developing diffuse gastric cancer 1-3 and lobular breast cancer 4-6 during life-time. CDH1 germline gene alterations (mutations or deletions), resulting in E-cadherin inactivation, are the only causative events described till now and were identified in approximately 30% of HDGC cases. 2,3,7 To date, 122 different germline mutations have been described in these families, 8 being the majority of them of the nonsense type, leading to alternative premature termination codons. 3 This type of CDH1 mutant transcripts is commonly downregulated by nonsensemediated decay leading to E-cadherin loss of function 9 and these patients are considered high-risk carriers and are counseled to perform prophylactic total gastrectomy. 10 In about 20% of HDGC families, carriers show CDH1 germline missense mutations 11 and, in contrast to truncating mutations, their pathogenic significance is not straightforward, therefore constituting a problem in terms of genetic counseling and surveillance.In 2004, Fitzgerald and Caldas 10 suggested that the significance of CDH1 missense mutations should be assessed in at least four affected members within a HDGC family in combination with functional and

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Section: Cdh1 Missense Mutations Affect E-cadherin Subcellular Localimentioning

confidence: 99%

Section: Cdh1 Missense Mutations Affect E-cadherin Subcellular Localimentioning

confidence: 99%

The importance of E-cadherin binding partners to evaluate the pathogenicity of E-cadherin missense mutations associated to HDGC

et al. 2012

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“…[21][22][23][24][25][26][27][28][29][30][31] The abovementioned observations, showing that 77% of the sporadic diffuse tumors displayed overexpression of v6-containing CD44 isoforms by immunohistochemistry, and that 4 of 10 cases showed a statistically significant increase of the v6/v7-encoding CD44 transcripts expression levels in the tumor (all of them of the diffuse type) compared with non-neoplastic adjacent mucosa, led us to explore a putative relationship between de novo expression of v6-containing CD44 isoforms and E-cadherin loss/aberrant expression.…”

Section: V6-containing Cd44 Isoforms Are Highly Expressed In Primary mentioning

confidence: 99%

“…Furthermore, we explored the association of CD44 and E-cadherin expression, as E-cadherin has a pivotal functional role in GC. [21][22][23][24][25][26][27][28][29][30][31] In hereditary diffuse GC (HDGC), neoplastic cells show absent/aberrant E-cadherin expression and invade the gastric wall either isolated or in poorly cohesive clusters. HDGC early lesions remain undetected with currently used diagnostic tools.…”

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De novo expression of CD44 variants in sporadic and hereditary gastric cancer

Cunha

Oliveira

Wen

et al. 2010

Laboratory Investigation

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CD44 is the major ubiquitously expressed cell surface receptor for hyaluronate. The CD44 gene encodes several protein isoforms due to extensive alternative splicing and post-translational modifications. Some of these CD44 variable isoforms have been foreseen as key players in malignant transformation and their expression is highly restricted and highly specific, unlike the canonical CD44 standard isoform. In this study, we aimed at dissecting the mRNA splicing pattern of CD44 in normal stomach and gastric cancer (GC) cell lines (n ¼ 9) using cloning and quantitative mRNA amplification assays. Moreover, we assessed the RNA levels and protein expression pattern of relevant splicing forms in distinct premalignant and malignant gastric lesions (sporadic (n ¼ 43) and hereditary (n ¼ 3) forms) using real-time RT-PCR and immunohistochemistry. We also explored the association of CD44 and E-cadherin expression by immunohistochemistry, as E-cadherin has a pivotal functional role in GC. We established the pattern of CD44 variant forms in normal stomach and gastric malignancy. We observed that although exon v6-containing isoforms were rarely expressed in normal gastric mucosa, they became increasingly expressed both in gastric premalignant (hyperplastic polyps, complete and incomplete intestinal metaplasia, low-and high-grade dysplasia) and malignant lesions (cell lines derived from GCs, primary sporadic GCs and hereditary diffuse GCs (HDGCs)). Moreover, we verified that whenever E-cadherin expression was absent, exon v6-containing CD44 isoforms were overexpressed. The lack of expression of CD44 isoforms containing exon v6 in the surface and foveolar epithelia of normal stomach and, its de novo expression in premalignant, as well as in sporadic and hereditary malignant lesions of the stomach, pinpoint CD44 v6-containg isoforms as potential biomarkers for early transformation of the gastric mucosa. Further, our results raise the hypothesis of using CD44v6 as a marker of early invasive intramucosal carcinoma in HDGC CDH1 mutation carriers that lack CDH1 expression in their tumors.

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“…16 However, these agents may introduce missense mutations that may be detrimental in cancer syndromes, by affecting protein structure or function. [17][18][19] In contrast, suppressor-tRNAs (sup-tRNAs) are able to induce stop codon readthrough by specifically introducing the cognate amino acid at the premature stop codon (PTC) site. 20 As each sup-tRNA can recognize and interact with one of the three stop codons amber (UAG), opal (UGA) and ochre (UAA), due to an alteration in its anticodon, 15,20 these agents have high stop codon specificity and also show higher nonsense suppression efficiency than other readthrough strategies.…”

Section: Cdh1mentioning

confidence: 99%

Rescue of wild-type E-cadherin expression from nonsense-mutated cancer cells by a suppressor-tRNA

et al. 2014

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Hereditary diffuse gastric cancer (HDGC) syndrome, although rare, is highly penetrant at an early age, and is severe and incurable because of ineffective screening tools and therapy. Approximately 45% of HDGC families carry germline CDH1/Ecadherin alterations, 20% of which are nonsense leading to premature protein truncation. Prophylactic gastrectomy is the only recommended approach for all asymptomatic CDH1 mutation carriers. Suppressor-tRNAs can replace premature stop codons (PTCs) with a cognate amino acid, inducing readthrough and generating full-length proteins. The use of suppressor-tRNAs in HDGC patients could therefore constitute a less invasive therapeutic option for nonsense mutation carriers, delaying the development of gastric cancer. Our analysis revealed that 23/108 (21.3%) of E-cadherin-mutant families carried nonsense mutations that could be potentially corrected by eight suppressor-tRNAs, and arginine was the most frequently affected amino acid. Using site-directed mutagenesis, we developed an arginine suppressor-tRNA vector to correct one HDGC nonsense mutation. E-cadherin-deficient cell lines were transfected with plasmids carrying simultaneously the suppressor-tRNA and wild-type or mutant CDH1 mini-genes. RT-PCR, western blot, immunofluorescence, flow cytometry and proximity ligation assay (PLA) were used to establish the model, and monitor mRNA and protein expression and function recovery from CDH1 vectors. Cells expressing a CDH1 mini-gene, carrying a nonsense mutation and the suppressor-tRNA, recovered full-length E-cadherin expression and its correct localization and incorporation into the adhesion complex. This is the first demonstration of functional recovery of a mutated causative gene in hereditary cancer by cognate amino acid replacement with suppressor-tRNAs. Of the HDGC families, 21.3% are candidates for correction with suppressor-tRNAs to potentially delay cancer onset.

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E-cadherin mutations and cell motility: A genotype–phenotype correlation

Cited by 65 publications

References 48 publications

The importance of E-cadherin binding partners to evaluate the pathogenicity of E-cadherin missense mutations associated to HDGC

The importance of E-cadherin binding partners to evaluate the pathogenicity of E-cadherin missense mutations associated to HDGC

De novo expression of CD44 variants in sporadic and hereditary gastric cancer

Rescue of wild-type E-cadherin expression from nonsense-mutated cancer cells by a suppressor-tRNA

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