Browsing by Author "Licastro, Danilo"
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- Mixed lineage kinase 3 gene mutations in mismatch repair deficient gastrointestinal tumours.Publication . Velho, Sérgia; Oliveira, Carla; Paredes, Joana; Sousa, Sónia; Leite, Marina; Matos, Paulo; Milanezi, Fernanda; Ribeiro, Ana Sofia; Mendes, Nuno; Licastro, Danilo; Karhu, Auli; Oliveira, Maria José; Ligtenberg, Marjolijn; Hamelin, Richard; Carneiro, Fátima; Lindblom, Annika; Peltomaki, Paivi; Castedo, Sérgio; Schwartz, Simó Jr; Jordan, Peter; Aaltonen, Lauri A.; Hofstra, Robert M.W.; Suriano, Gianpaolo; Stupka, Elia; Fialho, Arsenio M; Seruca, RaquelMixed lineage kinase 3 (MLK3) is a serine/threonine kinase, regulating MAPkinase signalling, in which cancer-associated mutations have never been reported. In this study, 174 primary gastrointestinal cancers (48 hereditary and 126 sporadic forms) and 7 colorectal cancer cell lines were screened for MLK3 mutations. MLK3 mutations were significantly associated with MSI phenotype in primary tumours (P = 0.0005), occurring in 21% of the MSI carcinomas. Most MLK3 somatic mutations identified were of the missense type (62.5%) and more than 80% of them affected evolutionarily conserved residues. A predictive 3D model points to the functional relevance of MLK3 missense mutations, which cluster in the kinase domain. Further, the model shows that most of the altered residues in the kinase domain probably affect MLK3 scaffold properties, instead of its kinase activity. MLK3 missense mutations showed transforming capacity in vitro and cells expressing the mutant gene were able to develop locally invasive tumours, when subcutaneously injected in nude mice. Interestingly, in primary tumours, MLK3 mutations occurred in KRAS and/or BRAF wild-type carcinomas, although not being mutually exclusive genetic events. In conclusion, we have demonstrated for the first time the presence of MLK3 mutations in cancer and its association to mismatch repair deficiency. Further, we demonstrated that MLK3 missense mutations found in MSI gastrointestinal carcinomas are functionally relevant.
- Transcription initiation arising from E-cadherin/CDH1 intron2: a novel protein isoform that increases gastric cancer cell invasion and angiogenesisPublication . Pinheiro, Hugo; Carvalho, Joana; Oliveira, Patrícia; Ferreira, Daniel; Teixeira Pinto, Marta; Osório, Hugo; Licastro, Danilo; Bordeira-Carriço, Renata; Jordan, Peter; Lazarevic, Dejan; Sanges, Remo; Stupka, Elia; Huntsman, David; Seruca, Raquel; Oliveira, CarlaDisruption of E-cadherin (CDH1 gene) expression, subcellular localization or function arises during initiation and progression of almost 90% of all epithelial carcinomas. Nevertheless, the mechanisms through which this occurs are largely unknown. Previous studies showed the importance of CDH1 intron 2 sequences for proper gene and protein expression, supporting these as E-cadherin cis-modulators. Through RACE and RT-PCR, we searched for transcription events arising from CDH1 intron 2 and discovered several new transcripts. One, named CDH1a, with high expression in spleen and absent from normal stomach, was demonstrated to be translated into a novel isoform, differing from canonical E-cadherin in its N-terminal, as determined by mass-spectrometry. Quantitative and functional assays showed that when overexpressed in an E-cadherin negative context, CDH1a replaced canonical protein interactions and functions. However, when co-expressed with canonical E-cadherin, CDH1a increased cell invasion and angiogenesis. Further, interferon-induced genes IFITM1 and IFI27 levels were increased upon CDH1a overexpression. Effects on invasion and IFITM1 and IFI27 expression were reverted upon CDH1a specific knockdown. Importantly, CDH1a was de novo expressed in gastric cancer cell lines. This study presents a new mechanism by which E-cadherin functions are impaired by cis-regulatory mechanisms possibly with the involvement of inflammatory machinery. If confirmed in other cancer models, our data encloses potential for designing targeted therapies to rescue E-cadherin function.