Percorrer por autor "Barros, Patrícia"
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- Activation of RAC1/PAK1 axis potentiates transcriptional upregulation of DNA damage response genes via the BCL6/STAT5 switchPublication . Barros, Patrícia; Amaral, Andreia; Abrantes, Leonor; Oliveira, Tiago; Lourio, Henriqueta; Silva, Maria João; Jordan, Peter; Gama-Carvalho, Margarida; Matos, PauloColorectal cancer is one of the most prevalent types of cancer worldwide. The GTPase RAC1 and its effector PAK1 have been found overexpressed or hyperactivated in colorectal cancers, particularly those with more aggressive and invasive features, leading to unfavourable clinical prognosis, often resulting from chemoresistance.
- Análise de coortes genómicas públicas de larga escala revelam BCL6 como marcador de prognóstico no cancro da mama Luminal APublication . Barros, Patrícia; Jordan, Peter; Matos, PauloO cancro da mama (CM) é a neoplasia maligna mais comum entre mulheres a nível mundial, constituindo uma das principais causas de mortalidade oncológica. Representa uma doença heterogénea, agrupada em subtipos moleculares com implicações prognósticas e terapêuticas distintas. O subtipo Luminal A é o mais prevalente e caracteriza-se por elevada expressão de recetores hormonais (estrogénio e progesterona), baixa taxa de proliferação e prognóstico geralmente favorável. No entanto, há evidências consistentes de risco significativo de recorrência tardia e de novos eventos neoplásicos, o que continua a levantar desafios na definição das estratégias de seguimento clínico. Neste estudo, analisámos dados genómicos da coorte de CM da base de dados pública TCGA (n=1247) para avaliar o valor prognóstico do gene BCL6, um regulador transcricional previamente implicado na progressão tumoral. Foram recolhidos dados de expressão de BCL6, subtipagem molecular (PAM50) e sobrevivência global (OS). Observou-se que, apesar da expressão de BCL6 estar globalmente diminuída nos tumores em comparação com o tecido normal, esta era significativamente mais elevada nos tumores Luminal A do que nos restantes subtipos, com um subgrupo (44%) a manter níveis semelhantes aos do tecido normal. Verificámos ainda que, exclusivamente neste subtipo, a expressão elevada de BCL6 se associava a pior sobrevivência (p=0,041). Estes resultados apontam para o potencial de BCL6 como biomarcador de estratificação de risco dentro do subtipo Luminal A, com possíveis implicações na definição da intensidade e duração do seguimento clínico a longo prazo
- B-Raf-induced senescence in colorectal cells is antagonized by expression of tumour-related Rac1bPublication . Henriques, Andreia; Barros, Patrícia; Moyer, Mary; Matos, Paulo; Jordan, PeterMutations in the BRAF oncogene have been identified as a tumour-initiating genetic event in mainly melanoma, thyroid and colon cancer, resulting in an initial proliferative stimulus that is followed by a growth arrest period known as oncogene-induced senescence (OIS). It remains unknown what triggers subsequent escape from OIS to allow further tumour progression. A previous analysis revealed that around 80% of colorectal tumours carrying a mutation in BRAF also overexpress splice variant Rac1b. We used normal NCM460 colonocytes as a model to express oncogenic B-Raf-V600E in the presence or absence of co-transfected Rac1b and analysed the effect on expression the senescence marker β-galactosidase and of the cell-cycle inhibitors p14, p15 and p21. We provide evidence that co-expression of splice variant Rac1b counteracts B-Raf-induced senescence. When oncogenic B-Raf-V600E was expressed we observed the induction of the senescence marker β-galactosidase and of the cell-cycle inhibitors p14, p15 and p21. Upon co-expression of splice variant Rac1b, the B-Raf-induced senescence was relieved and expression of the cell-cycle inhibitor proteins downregulated. Our data indicate the selection for increased Rac1b expression as one potential mechanism by which colorectal tumour cells can escape from B-Raf-induced OIS.
- BPA analogues affect intestinal barrier integrity and pro-inflammatory response in a coculture modelPublication . Pereira, Gonçalo; Barros, Patrícia; Matos, Paulo; Jordan, PeterBackground: Bisphenol (BP) A and its structural analogues are environmental pollutants with endocrine-disrupting properties and potential immune-modulating effects. Following legal restrictions on the use of BPA, structurally related compounds including BPAP, BPP, and BPS-MAE have been introduced as alternatives; however, their potential hazardous profiles remain largely unknown. Here we used a coculture cell model to investigate the effects of exposure to these BP analogues on intestinal barrier integrity, intestinal cell stress, and pro-inflammatory macrophage activation. Methods: As cellular model, Caco-2 cells were grown on filter membranes to a polarized epithelium-like layer, and cocultured with underlying basolateral THP-1 derived M0 macrophages. After apical exposure of the Caco-2 cells layer to BPs (0.1 -100 µM for 24 h), we determined transepithelial electrical resistance (TEER), polarized cell morphology by confocal microscopy, cellular stress markers (p-p38MAPK, p-JNK, p-eIF2α) by Western blot, and macrophage activation by IL-1β-transcript expression changes. In addition, M0-type macrophages were also directly incubated with BPA for comparison. Results: When M0 macrophages were directly exposed, BPA triggered IL-1β expression, an effect more evident after macrophage sensitization by the presence of interferon-γ. Apical exposure to BPA and BPS-MAE had little effect on TEER but induced some increase in epithelial stress markers, while BPAP and especially BPP clearly reduced TEER and polarized cell morphology, and showed a tendency to induce stress markers. In addition, apical cell exposure to BPP and BPS-MAE triggered clear expression of the pro-inflammatory marker IL-1β in sensitized M0 macrophages cocultured at the basolateral side, whereas BPAP and BPA were only effective at the highest concentration. Conclusion: Together, our data show that exposure of an intestinal epithelium-like layer to BPAP and BPS-MAE revealed adverse cellular effects similar to BPA, while BPP was clearly the most deleterious BP analogue. Pro-inflammatory macrophage activation was strongest after exposure to BPP followed by BPS-MAE.
- CFTR modulator drugs can reduce the invasive properties of colorectal cancer cellsPublication . Vicente, Luana; Barros, Patrícia; Gonçalves, Vânia; Oliveira, Paula; Jordan, Peter; Matos, PauloIntroduction: Colorectal cancer (CRC) remains a leading cause of cancer-related mortality, driven by complex genetic, epigenetic, and microenvironmental factors. Recent findings implicate the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel in CRC progression, as CFTR levels are notably reduced in sporadic CRCs, particularly in advanced and metastatic tumors, correlating with poorer patient outcomes. Additionally, cystic fibrosis (CF) patients, who carry CFTR mutations, have a 6-fold increased risk of early-onset CRC. Given recent advances in small-molecule modulators that restore CFTR function in CF patients, this study explored the potential of repositioning these modulators to address CFTR downregulation in sporadic CRC. Material and method: Using a panel of CRC cell lines, we investigated whether CFTR modulators can increase CFTR functional expression in cells with various genetic backgrounds and whether such improvements could reduce their oncogenic properties. Result and discussion: Our data show that treatment with the CFTR folding correctors VX-661 and VX-445 led to a significant, approximately three-fold increase in CFTR abundance in CRC cells expressing reduced but detectable levels of the channel. Additionally, these treatments significantly reduced the migratory and invasive behavior of Caco-2 and DLD-1 cells, particularly when combined with the CFTR potentiator VX-770. Our findings suggest that CFTR modulators may hinder the oncogenic properties of CRC cells. Further in vivo studies are necessary to fully assess their potential benefits for repositioning as a CRC treatment.
- Cystic Fibrosis modulator drugs inhibit migration of colorectal cancer cellsPublication . Vicente, Luana; Barros, Patrícia; Gonçalves, Vânia; Oliveira, Paula A.; Jordan, Peter; Matos, PauloColorectal cancer (CRC) remains a leading cause of cancer-related mortality, driven by complex genetic, epigenetic, and microenvironmental factors. Recent findings implicate the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel in CRC progression, as CFTR levels are notably reduced in sporadic CRCs, particularly in advanced and metastatic tumors, correlating with poorer patient outcomes. Additionally, cystic fibrosis (CF) patients, who carry CFTR mutations, have a 6-fold increased risk of early-onset CRC. Given recent advances in small-molecule modulators that restore CFTR function in CF patients, this study explored the potential of repositioning these modulators to address CFTR downregulation in sporadic CRC. Using a panel of CRC cell lines, we investigated whether CFTR modulators can increase CFTR functional expression in cells with various genetic backgrounds and whether such improvements could reduce their oncogenic properties. Our data show that treatment with the CFTR folding correctors VX-661 and VX-445 led to a significant, approximately three-fold increase in CFTR abundance in CRC cells expressing reduced but detectable levels of the channel. Additionally, these treatments significantly reduced the migratory and invasive behavior of Caco-2 and DLD-1 cells, particularly when combined with the CFTR potentiator VX-770. Our findings suggest that CFTR modulators may hinder the oncogenic properties of CRC cells. Further in vivo studies are necessary to fully assess their potential benefits for repositioning as a CRC treatment.
- Expression of tumor-related Rac1b antagonizes B-Raf-induced senescence in colorectal cellsPublication . Henriques, Andreia; Barros, Patrícia; Matos, Paulo; Jordan, PeterMutations in the BRAF oncogene have been identified as a tumor-initiating genetic event in mainly melanoma, thyroid and colon cancer, resulting in an initial proliferative stimulus that is followed by a growth arrest period known as oncogene-induced senescence (OIS). It remains unknown what triggers subsequent escape from OIS to allow further tumor progression. A previous analysis revealed that overexpression of splice variant Rac1b occurs in around 80% of colorectal tumors carrying a mutation in BRAF. Using both BRaf-V600E-directed RNAi and overexpression we demonstrate that this mutation does not directly lead to Rac1b overexpression, indicating the latter as an independent event during tumor progression. Nonetheless, we observed that expression of oncogenic BRaf-V600E in non-transformed colonocytes (NCM460 cell line) increased both the transcript and protein levels of p14ARF, p15INK4b and p21CIP1 and led to increased expression of β-galactosidase, all indicators of OIS induction. Interestingly, whereas the protein levels of these markers were reduced upon Rac1b overexpression, the levels of their respective transcripts remained unchanged. Importantly, the co-expression of Rac1b with B-Raf-V600E reverted the OIS phenotype, reducing the expression levels of the cell-cycle inhibitors and β-galactosidase to those of control cells. These data identify increased Rac1b expression as one potential mechanism by which colorectal tumor cells can escape from B-Raf-induced OIS.
- Expression of tumour-related Rac1b antagonizes B-Raf-induced senescence in colorectal cellsPublication . Henriques, Andreia FA; Barros, Patrícia; Moyer, Mary; Matos, Paulo; Jordan, PeterMutations in the BRAF oncogene have been identified as a tumor-initiating genetic event in mainly melanoma, thyroid and colon cancer, resulting in an initial proliferative stimulus that is followed by a growth arrest period known as oncogene-induced senescence (OIS). It remains unknown what triggers subsequent escape from OIS to allow further tumor progression. A previous analysis revealed that around 80% of colorectal tumors carrying a mutation in BRAF also overexpress splice variant Rac1b. We used normal NCM460 colonocytes as a model to express oncogenic B-Raf-V600E in the presence or absence of co-transfected Rac1b and then analyzed the effect on expression of senescence markers. When oncogenic B-Raf-V600E was expressed we observed the induction of the senescence-associated β-galactosidase and of the cell-cycle inhibitors p14, p15 and p21 whereas proliferation marker Ki67 was suppressed. Upon co-expression of splice variant Rac1b, but not of Rac1, the B-Raf-induced senescence phenotype was reverted and expression of the cell-cycle inhibitors downregulated in a reactive oxygen-species dependent manner. We thus provide evidence that co-expression of splice variant Rac1b counteracts B-Raf-induced senescence, indicating the selection for increased Rac1b expression as one potential mechanism by which colorectal tumor cells can escape from B-Raf-induced OIS.
- Impact of SHC-1 adaptor protein inhibitors on the plasma membrane abundance of the CFTR channel in epithelial cellsPublication . Barros, Patrícia; Pereira, Mariana F.L.; Tomilev, Alexey; Cortopassi, Gino; Jordan, Peter; Matos, PauloCFTR is a chloride and bicarbonate channel essential for ionic homeostasis in epithelial cells, and its plasma membrane (PM) abundance is often downregulated in chronic obstructive pulmonary disease (COPD) and colon cancer (CRC). CFTR endocytosis is promoted by Y512 phosphorylation by spleen tyrosine kinase, mediated by SHC-1, a phospho-tyrosine adaptor in MAPK signaling. Since the drug idebenone (IDE) disrupts SHC-1/phospho-insulin receptor interaction in hepatocytes, we tested its effects on CFTR internalization. In CFBE airway cells, IDE and a novel SHC-1 inhibitor (110#3) increased CFTR PM levels but also elevated PM levels of GLUT1 and E-cadherin, indicating a MAPK signaling-independent, non-specific action. Moreover, neither compound affected CFTR PM abundance or MAPK activity in 16HBE airway or Caco-2 intestinal cells. These findings underscore the context-dependent nature of SHC-1 signaling, suggesting IDE and related compounds may not universally disrupt SHC-1 interactions, or specifically block CFTR internalization in the airway or CRC cells.
- Inhibition of calpain 1 restores plasma membrane stability to pharmacologically rescued Phe508del-CFTR variantPublication . Matos, Ana M.; Pinto, Francisco R.; Barros, Patrícia; Amaral, Margarida D.; Pepperkok, Rainer; Matos, PauloCystic fibrosis (CF) is a genetic disease caused by mutations in the gene encoding CF transmembrane conductance regulator (CFTR), a chloride channel normally expressed at the surface of epithelial cells. The most frequent mutation, resulting in Phe-508 deletion, causes CFTR misfolding and its premature degradation. Low temperature or pharmacological correctors can partly rescue the Phe508del-CFTR processing defect and enhance trafficking of this channel variant to the plasma membrane (PM). Nevertheless, the rescued channels have an increased endocytosis rate, being quickly removed from the PM by the peripheral protein quality-control pathway. We previously reported that rescued Phe508del-CFTR (rPhe508del) can be retained at the cell surface by stimulating signaling pathways that coax the adaptor molecule ezrin (EZR) to tether rPhe508del-Na+/H+-exchange regulatory factor-1 complexes to the actin cytoskeleton, thereby averting the rapid internalization of this channel variant. However, the molecular basis for why rPhe508del fails to recruit active EZR to the PM remains elusive. Here, using a proteomics approach, we characterized and compared the core components of wt-CFTR- or rPhe508del-containing macromolecular complexes at the surface of human bronchial epithelial cells. We identified calpain 1 (CAPN1) as an exclusive rPhe508del interactor that prevents active EZR recruitment, impairs rPhe508del anchoring to actin, and reduces its stability in the PM. We show that either CAPN1 down-regulation or its chemical inhibition dramatically improves the functional rescue of Phe508del-CFTR in airway cells. These observations suggest that CAPN1 constitutes an appealing target for pharmacological intervention, as part of CF combination therapies restoring Phe508del-CFTR function.
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