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Papers by Paula Paulo

British Journal of Cancer

Theranostics

Rationale: Bladder cancer (BC) management demands the introduction of novel molecular targets for... more Rationale: Bladder cancer (BC) management demands the introduction of novel molecular targets for precision medicine. Cell surface glycoprotein CD44 has been widely studied as a potential biomarker of BC aggressiveness and cancer stem cells. However, significant alternative splicing and multiple glycosylation generate a myriad of glycoproteoforms with potentially distinct functional roles. The lack of tools for precise molecular characterization has led to conflicting results, delaying clinical applications. Addressing these limitations, we have interrogated the transcriptome and glycoproteome of a large BC patient cohort for splicing signatures. Methods: CD44 gene and its splicing variants were assessed by Real Time-Polymerase Chain Reaction (RT-PCR) and RNAseq in tumor tissues. The co-localization of CD44 and short O-glycans was evaluated by proximity ligation assay (PLA), immunohistochemistry and double-immunofluorescence. An innovative glycoproteogenomics approach, integrating transcriptomics-customized datasets and glycomics for protein annotation from nanoLC-ESI-MS/MS experiments, was developed and implemented to identify CD44 variants and associated glycosignatures. The impact of CD44 silencing on proliferation and invasion of BC cell lines and glycoengineered cells was determined by BrdU ELISA and Matrigel invasion assays, respectively. Antibody phosphoarrays were used to investigate the role of CD44 and its glycoforms in the activation of relevant oncogenic signaling pathways. Results: Transcriptomics analysis revealed remarkable CD44 isoforms heterogeneity in bladder cancer tissues, as well as associations between short CD44 standard splicing isoform (CD44s), invasion and poor prognosis. We further demonstrated that targeting short O-glycoforms such as the Tn and sialyl-Tn antigens was key to overcome the lack of cancer specificity presented by CD44. Glycoproteogenomics

Journal of Experimental & Clinical Cancer Research, 2021

Background Muscle invasive bladder cancer (MIBC) remains amongst the deadliest genitourinary mali... more Background Muscle invasive bladder cancer (MIBC) remains amongst the deadliest genitourinary malignancies due to treatment failure and extensive molecular heterogeneity, delaying effective targeted therapeutics. Hypoxia and nutrient deprivation, oversialylation and O-glycans shortening are salient features of aggressive tumours, creating cell surface glycoproteome fingerprints with theranostics potential. Methods A glycomics guided glycoproteomics workflow was employed to identify potentially targetable biomarkers using invasive bladder cancer cell models. The 5637 and T24 cells O-glycome was characterized by mass spectrometry (MS), and the obtained information was used to guide glycoproteomics experiments, combining sialidase, lectin affinity and bottom-up protein identification by nanoLC-ESI-MS/MS. Data was curated by a bioinformatics approach developed in-house, sorting clinically relevant molecular signatures based on Human Protein Atlas insights. Top-ranked targets and glycofor...

European Urology Oncology, 2021

Background: Germline ATM mutations are suggested to contribute to predisposition to prostate canc... more Background: Germline ATM mutations are suggested to contribute to predisposition to prostate cancer (PrCa). Previous studies have had inadequate power to estimate variant effect sizes. Objective: To precisely estimate the contribution of germline ATM mutations to PrCa risk. Design, setting, and participants: We analysed next-generation sequencing data from 13 PRACTICAL study groups comprising 5560 cases and 3353 controls of European ancestry. Outcome measurements and statistical analysis: Variant Call Format files were harmonised, annotated for rare ATM variants, and classified as tier 1 (likely pathogenic) or tier 2 (potentially deleterious). Associations with overall PrCa risk and clinical subtypes were estimated. Results and limitations: PrCa risk was higher in carriers of a tier 1 germline ATM variant, with an overall odds ratio (OR) of 4.4 (95% confidence interval [CI]: 2.0-9.5). There was also evidence that PrCa cases with younger age at diagnosis (<65 yr) had elevated tier 1 variant frequencies (p difference = 0.04). Tier 2 variants were also associated with PrCa risk, with an OR of 1.4 (95% CI: 1.1-1.7). Conclusions: Carriers of pathogenic ATM variants have an elevated risk of developing PrCa and are at an increased risk for earlier-onset disease presentation. These results provide information for counselling of men and their families. Patient summary: In this study, we estimated that men who inherit a likely pathogenic mutation in the ATM gene had an approximately a fourfold risk of developing prostate cancer. In addition, they are likely to develop the disease earlier.

Cancer Epidemiology, Biomarkers & Prevention, 2010

Background: A pressing clinical issue in prostate cancer is to distinguish which men will have an... more Background: A pressing clinical issue in prostate cancer is to distinguish which men will have an indolent or aggressive course of disease. Clinical variables such as Gleason grade and stage are useful predictors of lethal cancer; however, the low predictive values of the common Gleason scores, changes in grading over time, and earlier diagnosis of patients due to screening limits their clinical utility. Identifying genetic variants associated with lethal prostate cancer could inform clinical decision making. Methods: We conducted a genome-wide association study, comparing lethal prostate cancer cases to cases surviving at least 10 years beyond their initial diagnosis. Genotyping was done with the Affymetrix 5.0 chip [∼500,000 single nucleotide polymorphisms (SNP) and 1,483 copy number variants (CNV)] on DNA from participants in the Physicians' Health Study and Health Professionals Follow-up Study (196 lethal cases, 368 long-term survivors). After excluding SNPs and individuals ...

Nature communications, Nov 5, 2018

Chromosome 8q24 is a susceptibility locus for multiple cancers, including prostate cancer. Here w... more Chromosome 8q24 is a susceptibility locus for multiple cancers, including prostate cancer. Here we combine genetic data across the 8q24 susceptibility region from 71,535 prostate cancer cases and 52,935 controls of European ancestry to define the overall contribution of germline variation at 8q24 to prostate cancer risk. We identify 12 independent risk signals for prostate cancer (p < 4.28 × 10), including three risk variants that have yet to be reported. From a polygenic risk score (PRS) model, derived to assess the cumulative effect of risk variants at 8q24, men in the top 1% of the PRS have a 4-fold (95%CI = 3.62-4.40) greater risk compared to the population average. These 12 variants account for ~25% of what can be currently explained of the familial risk of prostate cancer by known genetic risk factors. These findings highlight the overwhelming contribution of germline variation at 8q24 on prostate cancer risk which has implications for population risk stratification.

Background: Gastric carcinoma is the second leading cause of cancer-associated death worldwide. T... more Background: Gastric carcinoma is the second leading cause of cancer-associated death worldwide. The high mortality associated with this disease is in part due to limited knowledge about gastric carcinogenesis and a lack of available therapeutic and prevention strategies. MUC1 is a high molecular weight transmembrane mucin protein expressed at the apical surface of most glandular epithelial cells and a major component of the mucus layer above gastric mucosa. Overexpression of MUC1 is found in approximately 95 % of human adenocarcinomas, where it is associated with oncogenic activity. The role of MUC1 in gastric cancer progression remains to be clarified. Methodology: We downregulated MUC1 expression in a gastric carcinoma cell line by RNA interference and studied the effects on cellular proliferation (MTT assay), apoptosis (TUNEL assay), migration (migration assay), invasion (invasion assay) and aggregation (aggregation assay). Global gene expression was evaluated by microarray analy...

Cancers, 2020

The identification of recurrent founder variants in cancer predisposing genes may have important ... more The identification of recurrent founder variants in cancer predisposing genes may have important implications for implementing cost-effective targeted genetic screening strategies. In this study, we evaluated the prevalence and relative risk of the CHEK2 recurrent variant c.349A>G in a series of 462 Portuguese patients with early-onset and/or familial/hereditary prostate cancer (PrCa), as well as in the large multicentre PRACTICAL case–control study comprising 55,162 prostate cancer cases and 36,147 controls. Additionally, we investigated the potential shared ancestry of the carriers by performing identity-by-descent, haplotype and age estimation analyses using high-density SNP data from 70 variant carriers belonging to 11 different populations included in the PRACTICAL consortium. The CHEK2 missense variant c.349A>G was found significantly associated with an increased risk for PrCa (OR 1.9; 95% CI: 1.1–3.2). A shared haplotype flanking the variant in all carriers was identifi...

Bladder cancer (BC) management demands the introduction of novel molecular targets for precision ... more Bladder cancer (BC) management demands the introduction of novel molecular targets for precision medicine. Cell surface glycoprotein CD44 has been widely studied as a potential biomarker of BC aggressiveness and cancer stem cells. However, significant alternative splicing and multiple glycosylation generate a myriad of glycoproteoforms with potentially distinct functional roles. The lack of tools for precise molecular characterization has led to conflicting results, delaying clinical applications. Addressing these limitations, we have interrogated the transcriptome of a large BC patient cohort for splicing signatures. Remarkable CD44 heterogeneity was observed, as well as associations between short CD44 standard splicing isoform (CD44s), invasion and poor prognosis. In parallel, immunoassays showed that targeting short O-glycoforms could hold the key to improve CD44 cancer specificity. This prompted the development of a glycoproteogenomics approach, building on the integration of tr...

Bladder cancer constitutes one of the deadliest genitourinary diseases, especially when diagnosed... more Bladder cancer constitutes one of the deadliest genitourinary diseases, especially when diagnosed at late stages. These tumours harbour microenvironmental niches characterized by low levels of oxygen (hypoxia) and limited glucose supply due to poor vascularization. However, the synergic contribution of these features to disease development is poorly understood. Here, we demonstrated that cells with distinct histopathological and molecular backgrounds responded similarly to such stimuli. Cancer cells arrested proliferation, significantly increased invasive capacity in vitro and enhanced tolerance to cisplatin-based chemotherapy. Reoxygenation and access to glucose restored basal proliferation and invasion levels without triggering stress-induced apoptosis, denoting significant cellular plasticity in adapting to microenvironmental cues. Whole transcriptomics showed major molecular reprogramming, supporting main functional alterations. Metabolomics evidenced fatty acids β-oxidation as ...

Cancer Epidemiology Biomarkers & Prevention, 2020

International Journal of Molecular Sciences, 2020

Prostate cancer (PrCa) ranks among the top five cancers for both incidence and mortality worldwid... more Prostate cancer (PrCa) ranks among the top five cancers for both incidence and mortality worldwide. A significant proportion of PrCa susceptibility has been attributed to inherited predisposition, with 10–20% of cases expected to occur in a hereditary/familial context. Advances in DNA sequencing technologies have uncovered several moderate- to high-penetrance PrCa susceptibility genes, most of which have previously been related to known hereditary cancer syndromes, namely the hereditary breast and ovarian cancer (BRCA1, BRCA2, ATM, CHEK2, and PALB2) and Lynch syndrome (MLH1, MSH2, MSH6, and PMS2) genes. Additional candidate genes have also been suggested, but further evidence is needed to include them in routine genetic testing. Recommendations based on clinical features, family history, and ethnicity have been established for more cost-efficient genetic testing of patients and families who may be at an increased risk of developing PrCa. The identification of alterations in PrCa pre...

We aimed to determine the effect of sample size on performance of polygenic hazard score (PHS) mo... more We aimed to determine the effect of sample size on performance of polygenic hazard score (PHS) models in predicting the age at onset of prostate cancer. Age and genotypes were obtained for 40,861 men from the PRACTICAL consortium. The dataset included 201,590 SNPs per subject, and was split into training (34,444 samples) and testing (6,417 samples) sets. Two PHS model-building strategies were investigated. Established-SNP model considered 65 SNPs that had been associated with prostate cancer in the literature. A stepwise SNP selection was used to develop Discovery-SNP models. The performance of each PHS model was calculated for random sizes of the training set (1 to 30 thousand). The performance of a representative Established-SNP model was estimated for random sizes of the testing set (0.5 to 6 thousand). Mean HR98/50 (hazard ratio of top 2% to the average in the test set) of the Established-SNP model increased from 1.73[95%CI: 1.69-1.77] to 2.41[2.40-2.43] when the number of train...

Background: Genetic risk stratification may inform decisions of whether, and when, a man should u... more Background: Genetic risk stratification may inform decisions of whether, and when, a man should undergo prostate cancer (PCa) screening. We previously validated a polygenic hazard score (PHS), a weighted sum of 54 single-nucleotide polymorphism genotypes, for accurate prediction of age of onset of aggressive PCa and improved screening performance. We now assess the potential impact of PHS-informed screening. Methods: United Kingdom population data were fit to a continuous model of age-specific PCa incidence. Using hazard ratios estimated from ProtecT trial data, age-specific incidence rates were calculated for percentiles of genetic risk. Incidence of higher-grade PCa (Gleason≥7) was estimated from age-specific data from the linked CAP trial. PHS and incidence data were combined to give a risk-equivalent age, when a man with a given PHS percentile will have risk of higher-grade PCa equivalent to that of a typical man at age 50 (50-years standard). Positive predictive value (PPV) of ...

Nature Genetics, 2019

In the version of this article initially published, the name of author Manuela Gago-Dominguez was... more In the version of this article initially published, the name of author Manuela Gago-Dominguez was misspelled as Manuela Gago Dominguez. The error has been corrected in the HTML and PDF version of the article.

Nature genetics, Jan 11, 2018

Genome-wide association studies (GWAS) and fine-mapping efforts to date have identified more than... more Genome-wide association studies (GWAS) and fine-mapping efforts to date have identified more than 100 prostate cancer (PrCa)-susceptibility loci. We meta-analyzed genotype data from a custom high-density array of 46,939 PrCa cases and 27,910 controls of European ancestry with previously genotyped data of 32,255 PrCa cases and 33,202 controls of European ancestry. Our analysis identified 62 novel loci associated (P < 5.0 × 10) with PrCa and one locus significantly associated with early-onset PrCa (≤55 years). Our findings include missense variants rs1800057 (odds ratio (OR) = 1.16; P = 8.2 × 10; G>C, p.Pro1054Arg) in ATM and rs2066827 (OR = 1.06; P = 2.3 × 10; T>G, p.Val109Gly) in CDKN1B. The combination of all loci captured 28.4% of the PrCa familial relative risk, and a polygenic risk score conferred an elevated PrCa risk for men in the ninetieth to ninety-ninth percentiles (relative risk = 2.69; 95% confidence interval (CI): 2.55-2.82) and first percentile (relative risk ...

Nature communications, Jun 11, 2018

Prostate cancer is a polygenic disease with a large heritable component. A number of common, low-... more Prostate cancer is a polygenic disease with a large heritable component. A number of common, low-penetrance prostate cancer risk loci have been identified through GWAS. Here we apply the Bayesian multivariate variable selection algorithm JAM to fine-map 84 prostate cancer susceptibility loci, using summary data from a large European ancestry meta-analysis. We observe evidence for multiple independent signals at 12 regions and 99 risk signals overall. Only 15 original GWAS tag SNPs remain among the catalogue of candidate variants identified; the remainder are replaced by more likely candidates. Biological annotation of our credible set of variants indicates significant enrichment within promoter and enhancer elements, and transcription factor-binding sites, including AR, ERG and FOXA1. In 40 regions at least one variant is colocalised with an eQTL in prostate cancer tissue. The refined set of candidate variants substantially increase the proportion of familial relative risk explained...

BMJ (Clinical research ed.), Jan 10, 2018

To develop and validate a genetic tool to predict age of onset of aggressive prostate cancer (PCa... more To develop and validate a genetic tool to predict age of onset of aggressive prostate cancer (PCa) and to guide decisions of who to screen and at what age. Analysis of genotype, PCa status, and age to select single nucleotide polymorphisms (SNPs) associated with diagnosis. These polymorphisms were incorporated into a survival analysis to estimate their effects on age at diagnosis of aggressive PCa (that is, not eligible for surveillance according to National Comprehensive Cancer Network guidelines; any of Gleason score ≥7, stage T3-T4, PSA (prostate specific antigen) concentration ≥10 ng/L, nodal metastasis, distant metastasis). The resulting polygenic hazard score is an assessment of individual genetic risk. The final model was applied to an independent dataset containing genotype and PSA screening data. The hazard score was calculated for these men to test prediction of survival free from PCa. Multiple institutions that were members of international PRACTICAL consortium. All conso...