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- 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.
- 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.
- 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.
- Regulation of the alternative splicing of RAC1B in tumor cellsPublication . Bizarro, Inês; Jordan, Peter; Gonçalves, VâniaA subgroup of colon tumors is characterized by the simultaneous presence of an oncogenic mutation in BRAF and overexpression of RAC1B, a splicing variant of the GTPase RAC1. RAC1B overexpression has also been identified in pancreatic, breast, lung and thyroid cancer. Inclusion of alternative exon 3b originates variant protein RAC1B and depends on two splicing factors, SRSF1 and SRSF3, which promote and inhibit, respectively, inclusion of this exon in colorectal cancer cells. ESRP1 has also been found to promote RAC1B expression in these cells. Here we studied whether these 3 splicing factors are also modulators of alternative splicing of RAC1B in breast and lung cancer cells. A similar role for SRSF1 and SRSF3 was found in breast cancer cells. Intriguingly, ESRP1 revealed an opposite role in alternative splicing of RAC1B comparing to what was previously observed in colorectal cancer cells. Currently, we are extending this study to lung cancer cells.
- 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.
- Loss of BCL6 transcriptional repressor impacts migration but not viability in MCF-7 breast cancer cellsPublication . Dyson-Jorge, João; Jordan, Peter; Barros, Patrícia; Matos, PauloBreast cancer (BC) incidence has risen over the past two decades, now being the most prevalent cancer worldwide and the second leading cause of cancer-related deaths. Despite advancements in BC treatment, challenges like acquired resistance, recurrence, and metastasis persist. BCL6, a transcriptional repressor, acts as an oncogene in BC, being downregulated in about half of primary tumors of all BC subtypes and associated with disease progression and poor patient prognosis. This underscores the need to better understand BCL6 role in BC development. Therefore, we used RNA interference to explore the impact of BCL6 depletion on the oncogenic progression of MCF-7 cells, a low-tumorigenic estrogen receptor-positive cell line. While BCL6 is known to regulate normal mammary cell proliferation and differentiation, its depletion did not affect MCF-7 cell proliferation or viability but significantly reduced their individual and collective migratory properties. RNA microarray analysis identified a set of genes upregulated following BCL6 depletion, including S100A7, previously reported to inhibit MCF-7 cell migration and invasion by reducing MMP9 secretion. However, our findings showed that S100A7 downregulation alone did not affect MCF-7 migration. Moreover, simultaneous depletion of BCL6 and S100A7 failed to restore MCF-7 cell migratory behavior.
- Multiple non-contiguous interstitial deletions in 5q21q22.1, including the CHD1 gene, identified in a boy with developmental delay and severe language impairmentPublication . Marques, Bárbara; Pedro, Sónia; Serafim, Sílvia; Tarelho, Ana Rita; Ferreira, Cristina; Catanho, Joana Adelaide; Moreira, Ana; Carvalho, Inês; Correia, HildebertoIntellectual disability (ID), developmental delay (DD), and behavioural disorders are complex neurodevelopmental conditions associated with multifactorial etiologies, including genetic factors. Chromosomal microarray analysis (CMA) is a valuable tool in identifying copy number variations (CNVs) contributing to neurodevelopmental disorders. Here we report a 4-year-old male with DD, severe language impairment, behavioral disturbances, macrocephaly, facial dysmorphisms, and delay walking (at 20 months). He was born to nonconsanguineous parents. Family history is significant for maternal intellectual disability and paternal neonatal hypoxic-ischemic encephalopathy, which resulted in hemiparesis and language impairment. CMA revealed three heterozygous interstitial deletions: 2.12Mb at 5q15q21.1(97929163-100045362), encompassing the CHD1 gene; 684Kb at 5q21.3(104580978-105264711), a gene-free region; and 3.69Mb at 5q21.3q22.1(107047547-110727429), including the SLC25A46 gene. Parental segregation studies revealed that all three deletions were maternally inherited. Although the identified non-contiguous losses may suggest a complex chromosomal rearrangement (CCR), no further studies were performed. Interstitial deletions of the middle region of the long arm of chromosome 5 are rare, and cases of CCR involving only this chromosome are even more rare. Most of the CCR are associated to intellectual disability. Missense variants in CHD1 have been associated with Pilarowski-Bjornsson syndrome, a neurodevelopmental disorder characterized by ID, DD, dysmorphic features, and apraxia of speech. However, deletion involving this gene are rare and may lead to speech abnormalities in the absence of intellectual disability (ID) or other major neurodevelopmental disorders. This phenotypic variability suggests that both CHD1 deletions and missense variants may exhibit variable expressivity or incomplete penetrance. This case highlights a possible link between CHD1 deletion and an autosomal dominant complex neurodevelopmental disorder and the diagnostic utility of cytogenetic studies in identifying complex genetic etiologies underlying CCR.
- Loss of function of PCDH11X gene in the pathogenesis of neurodevelopmental disordersPublication . Pedro, Sónia; Marques, Bárbara; Tarelho, Ana Rita; Vicente, M. Lurdes; Correia, HildebertoIntroduction: The PCDH11X gene, predominantly expressed in the brain, plays an important role in cell–cell communication, dendritic synaptic plasticity, cerebral lateralization, and verbal ability. Although it is not currently classified as a morbid gene, loss-of-function (LoF) variants in PCDH11X have been reported in the literature as having a moderate association whit autism spectrum disorder (ASD). These variants have been described exclusively in males with ASD, and a recent X-chromosome-wide association study highlighted significant associations between intronic and intergenic variants in this locus and ASD in males. We report a 6-year-old male patient presenting a complex neurodevelopmental phenotype, including learning difficulties, mild motor delay, speech sound disorder, selective mutism, and global hypotonia, as well as facial dysmorphisms, such on prominent large ears, midface hypoplasia, and a high forehead. Methodology: DNA was extracted from blood sample and chromosomal microarray analysis (CMA), was performed using Thermofisher CytoScan HD. Results: CMA profile identified a 2.80 Mb interstitial deletion at Xq21.31q21.32 (arr[GRCh37] Xq21.31q21.32(90,594,030_93,397,746)x0). Discussion: The interstitial deletion identified in this patient encompasses three OMIM listed genes, PABPC5, PCDH11X, and NAP1L3, none of which are currently classified as morbid and being associated to disease. However, the patient’s phenotype, characterized by neurodevelopmental disorders, aligns with previous reports linking PCDH11X alterations to neurodevelopmental impairments. Although the current level of evidence is considered moderate, this case suggesting a potential role for PCDH11X loss of function in the pathogenesis of neurodevelopmental disorders. In particular, the overlap between the clinical features observed and the known functions of PCDH11X, especially those related to language and communication, reinforces the hypothesis that this gene, despite not being classified as morbid to date, may contribute causally to such phenotypes when disrupted.
- Prenatal Diagnosis in a Fetus with Fetal Growth Restriction and a 4-Copy Gain in the 15q11.2q13.1 RegionPublication . Simão, Laurentino R.; Marques, Bárbara S.; Pedro, Sónia I.; Serafim, Sílvia S.; Alves, Ana C.; Tarelho, Ana R.; Ferreira, Cristina M.; Silva, Marisa D.; Peliano, Ricardo C.; Oliveira, Nayara D.; Brito, Filomena T.; Viegas, Mónica D.; Carvalho, Inês S.; Bernardeco, Joana S.; Cohen, Álvaro E.; Correia, Hildeberto O.Fetal growth restriction (FGR) is a common ultrasound finding in pregnancy that can result from maternal, fetal, placental, environmental factors, or their interaction, and its diagnosis is based on ultrasound screening. This finding is associated with a significant increase in perinatal morbidity and mortality. On the other hand, copy number variants (CNV) in the 15q11.2q13.1 region are associated with recurrent microdeletion/microduplication syndromes, in which the phenotype depends on the parental origin of the CNV. Here we report on a foetus from a healthy 32-year-old woman with early RCF in prenatal screening. A chromosomal microarray analysis (CMA) was requested, revealing a pathogenic gain of 4 copies, with 5.77 Mb, corresponding to the justacentromeric region 15q11.2q13.1. The karyotype was established as mos 47,XX,+mar dn[40]/46,XX[28]. As the outcome of this CNV depended on the parental origin of the affected allele, MS-MLPA was performed, which showed that it was inherited maternally. The detected CNV is a recurrent known microduplication witch phenotype is dependent on the parental origin of the duplication. When it has maternally origin it has a severe outcome with hypotonia, cognitive deficit, language delay, behavioral disorders, seizures, among others. If the CNV has paternal origin, although some patients might show developmental delays and behavioral disturbances, most cases are rarely symptomatic. In prenatal diagnosis, few cases have been described in the literature, with intrauterine growth restriction being one of the ultrasound abnormalities reported when the abnormality is of maternal origin. This case reinforces the importance of the combined use of cytogenetic and molecular cytogenetic technologies, such as karyotyping, MS-MLPA, and CMA, which play a key role in identifying the origins and genetic makeup of sSMC, which would contribute significantly to genetic counselling in prenatal diagnosis.
- Genetic Analysis of Early and Recurrent Pregnancy Loss: Challenges and AdvancesPublication . Ferreira, Cristina; Tarelho, Ana Rita; Pedro, Sónia; Marques, Bárbara; Viegas, Mónica; Correia, HildebertoPregnancy loss is defined as the spontaneous termination of a pregnancy before fetal viability and affects approximately one in four pregnancies. Genetic analysis of early or recurrent fetal losses is crucial for elucidating the underlying causes of miscarriage, thereby guiding clinical management and providing prognostic information to affected couples. Genetic anomalies, particularly chromosomal abnormalities, are implicated in approximately 50% to 70% of spontaneous abortions in the first trimester. Identifying these anomalies aids in understanding the etiology of pregnancy loss and offers valuable insights for future reproductive planning. Various sample types and genetic testing methodologies are employed in this context, each with distinct advantages and limitations. Traditional cytogenetic analysis, such as karyotyping, requires viable fetal cells obtained through culture. However, this method has a success rate of only about 58% due to potential culture failures and maternal cell contamination, which can lead to inconclusive or misleading results. In contrast, molecular techniques like chromosomal microarray analysis (CMA) do not rely on cell culture, offering higher resolution in detecting chromosomal abnormalities, improving diagnostic yield, and reducing the incidence of inconclusive results. However, CMA requires a sufficient amount of fetal DNA, which may be difficult to obtain due to maternal contamination or the challenge of collecting the conceptus product. The use of cell-free fetal DNA (cffDNA) from maternal blood has emerged as a promising method for evaluating fetal ploidy status, although large-scale validation of this approach is still required. In this study, we present our laboratory's experience in analyzing different sample types and employing various methodologies to investigate pregnancy loss. Our findings contribute to the ongoing discussion on optimizing genetic diagnostic strategies for early and recurrent pregnancy loss, improving patient outcomes, and informing future research directions.