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- Analysis and evaluation of genotoxicity and carcinogenicity assessment in EU legislation to improve regulatory implementation of NAMs: A focus on in silico approachesPublication . Bossa, Cecília; Raitano, Giuseppa; Benfenati, Emilio; Alivernini, Silvia; Andreoli, Cristina; Aquilina, G.; Attias, L.; Dusinska, Maria; El Yamani, N.; Louro, Henriqueta; Marcon, Francesca; Rundèn-Pran, E.; Russo, Maria Teresa; Silva, Maria João; Battistelli, Chiara LauraGenotoxicity and carcinogenicity are key endpoints for the risk assessment of all types of substances. Research on alternatives to animal testing for these endpoints has been active for decades, leading to the development of short-term in vitro tests that are integrated into current testing strategies. Nevertheless, high relevance is still devoted to data from in vivo studies. In parallel, progress in the comprehension of mechanisms underpinning genotoxicity and genotoxic carcinogenicity processes, together with the analysis of the great wealth of experimental data produced, allowed the discovery of structural determinants utilized in quantitative and qualitative structure-activity relationships and enabling in silico predictions of these endpoints. Presented here is a case study part of the collective effort carried out within the European Partnership for the Assessment of Risks from Chemicals (PARC) to address the challenges associated with innovation in chemical risk assessment, including the phasing out of animal testing through the introduction of New Approach Methodologies (NAMs) [1,2]. The case study aims to analyze current practices of the regulatory evaluation of genotoxicity and carcinogenicity hazard in several EU frameworks, in order to highlight needs and challenges in the actual or potential use of NAMs as well as short-and long- term goals towards the overcoming of animal testing. Among other NAMs, we are focusing on the role of in silico approaches highlighting strategies to increase the regulatory application and acceptance of QSAR based approaches. To this aim, the OECD QSAR Assessment Framework [3,4] has been identified as a suitable tool for evaluating the models and their predictions and will be applied to selected case studies. Moreover, a list of human relevant carcinogens has been developed as reference chemicals to evaluate and possibly refine in silico methodologies supporting a human-centric paradigm shift in toxicology.
- Dissecting the DIS3L2 target-specificity of transcripts committed to nonsense-mediated decay in human cellsPublication . Garcia-Moreno, Juan F.; Carvalho, Miguel P.; Lacerda, Rafaela; Romão, LuísaNonsense-mediated mRNA decay (NMD) is a conserved surveillance mechanism that eliminates mRNAs harboring premature termination codons (PTCs) and regulates the expression of certain physiological transcripts. The 3’-to-5’ exoribonuclease DIS3L2 degrades different RNAs independently of the RNA exosome, following uridylation at the 3' end by the terminal uridylyl transferases TUT4 and TUT7. We and others have shown that DIS3L2 is involved in NMD in an uridylation-dependent manner, being its function in NMD target-specific (Kurosaki et al. 2018; da Costa et al. 2019). Now, we aim to characterize the mechanisms involved in DIS3L2/NMD-target specificity. We used our RNA-seq data already obtained and validated and compared the transcripts upregulated upon DIS3L2 knockdown (REF) with a validated NMD-target set (Colombo et al., 2017). We observed that about 7% of DIS3L2-sensitive transcripts overlap with known NMD-targets. Considering the different groups of transcripts, we then analyzed specific features that make some NMD-targets sensitive to DIS3L2 (so called DIS3L2/NMD-targets; group 1), versus the remaining NMD-targets (DIS3L2-resistant NMD-targets; group 2), the remaining DIS3L2-sensitive targets (group 3), or the remaining transcriptome (DIS3L2-resistant NMD-targets plus NMD-resistant transcriptome; group 4). We assessed the following genomic features: 5’ and 3’ untranslated region (UTR) lengths, 3’UTR GC-, AU-, G-, C-, A-, and U-contents, presence of 5’UTR upstream open reading frames (uORFs), and 3’UTR introns. Elevated G-, C-, and GC-contents in the 3’UTRs were the most consistent features distinguishing DIS3L2/NMD-targets from the group 4. Comparison between group 1 and 2, and 1 and 3 was not significant. To better characterize the importance of each transcript portion, we are also analyzing hybrid constructs combining regions of the DIS3L2/NMD-resistant human β-globin (HBB) gene and the DIS3L2/NMD-sensitive GADD45A gene expressed in DIS3L2 depleted cultured cells.
- Dysregulated gene expression in colorectal cancer upon exposure to bisphenol A alternatives - a new approachPublication . Lacerda, Rafaela; Ventura, Célia; Louro, Henriqueta; Silva, Maria João; Romão, LuísaBisphenol A (BPA) has been widely used in plastics and resins since the 1950s, making it a common part of everyday products like food containers and bottle linings. Alternative substances are increasingly replacing BPA, but they are raising health and environmental concerns. Some mimic BPA’s endocrine-disrupting effects, while others affect different biological pathways. Substitutions in bisphenols can alter their biological properties, including nuclear receptor activation. Some BPA alternatives, like BPS, BPF and BPZ, may also pose cancer risks by activating oestrogen receptors, potentially even more than BPA itself. They may also contribute to colorectal cancer (CRC). Research suggests that BPA and its substitutes can influence cancer progression by altering cellular pathways, promoting metastasis and affecting gene expression. One of the key steps in gene expression regulation is translation initiation, whose canonical pathway is globally impaired under stress conditions, like exposure to BPA alternatives. Thus, we will subject NCM460 (normal intestinal mucosa) and HCT116 (colorectal carcinoma) cells to BPS, BPF and BPZ exposure and identify the transcripts actively being translated in such conditions, using ribosome profiling. We will analyse data with the R package anota2seq and evaluate the positively identified targets (compared to total RNA sequencing) for the existence of alternative mechanisms of translation initiation regulating their expression. The accurate characterisation of such mechanisms will be crucial for designing antisense RNA oligomers (ASOs) for potential therapeutic approaches. We will evaluate the cytotoxic effects of BPS, BPF and BPZ in the presence or absence of the selected alternatively translated transcripts (functional or targeted with the designed ASOs). Cytotoxic effects will be assessed through in vitro assays, analysing metabolic activity, membrane integrity, and cell proliferation. Thus, our research explores protein synthesis dysregulation to reduce CRC risks from BPS, BPF and BPZ exposure — an emerging public health issue.
- The potential function of alternative translation initiation of Argonaute 1 in cancerPublication . Vieira da Silva, Verónica; Lacerda, Rafaela; Romão, LuísaTranslation is one of the most regulated and energy-consuming cellular processes crucial for proper cell function. Translation is initiated by the canonical cap-dependent mechanism. However, under stress conditions, the initiation of canonical translation is inhibited, which allows the translation of specific proteins via alternative mechanisms. This project aims to understand the biological relevance of alternative protein synthesis mechanisms in Argonaute 1 (AGO1) expression. The AGO1 protein is involved in microRNA regulation, gene expression modulation and inhibition. AGO1 is also involved in the regulation of gene expression by RNA interference (RNAi), and its deregulation can lead to the activation of oncogenes or the suppression of tumor suppressor genes, contributing to the development and progression of cancer. Our work has shown that AGO1 mRNA can be translated through a cap-independent initiation mechanism. An upstream open reading frame (uORF) has also been identified in its 5’ untranslated region (5’UTR), which may play a role in the initiation of AGO1 translation. The results showed that the 5’UTR of human AGO1 supports a cap-independent mechanism of translation initiation, which is maintained under stress conditions. However, our analyses did not provide conclusive evidence for a regulatory role of the uORF in this initiation process. To this end, the 5’UTR of human AGO1 was cloned into a bicistronic vector containing Renilla (RLuc) and Firefly luciferase (FLuc), with FLuc positioned downstream of the 5’UTR. Luminometry assays will be used to evaluate the relative FLuc/RLuc translation efficiency under the control of the AGO1 5’UTR. The same approach will be used with monocistronistic reporter vectors, contaning only FLuc. This project aims to understand how these alternative mechanisms of mRNA translation initiation can influence AGO1 expression and help explain their potential roles in certain pathologies and cancer progression, such as colorectal cancer.
- The role of UPF1 cap-independent translation in colorectal cancerPublication . Lacerda, Rafaela; Menezes,Juliane; Elias, Adriana; Sousa, Sofia de; Romão, LuísaColorectal cancer (CRC) is one of the deadliest diseases worldwide with projections pointing towards an increase for the next two decades. Translation dysregulation of many genes contributes to CRC development, and here we are studying the role of translation dysregulation of up-frameshift 1 (UPF1) in CRC. This protein is involved in many cellular mechanisms such as nonsense-mediated mRNA decay, cell cycle progression, or telomere maintenance and homeostasis. It also works as a tumour suppressor protein in most cancers but not in CRC, in which UPF1 plays an oncogenic role. We used the Xena platform to perform in silico analyses that revealed UPF1 protein overexpressed in CRC, contrary to several other analysed cancers. Besides, UPF1 protein levels are increased in CRC compared to the counterpart normal tissues. Experimentally, we confirmed that UPF1 protein expression is maintained in different CRC cell lines under normal conditions or endoplasmic reticulum (ER) stress. To understand the mechanism underlying such maintenance, we used a bicistronic reporter construct to test whether UPF1 5’ untranslated region (UTR) can mediate alternative translation initiation and we concluded that such sequence drives cap-independent translation initiation, in both normal and stress conditions. Deletional and mutational analyses of UPF1 5’UTR showed that nucleotides 1–100 [stem-loop (SL) I] and 151–275 (SL III) — out of 275 nucleotides — are the minimal required sequences for the cap-independent translation initiation mechanism to work properly. Using RNA antisense oligonucleotides (ASOs) targeting UPF1 SL I and III, we observed a reduced UPF1 expression in HCT116 (CRC) cells, supporting the functional role of SL I and SL III in mediating cap-independent translation. Altogether, these results highlight the importance of cap-independent translation initiation in UPF1 expression regulation, in conditions that mimic the tumour microenvironment, and this might be used as a therapeutic target.
- Upstream Open Reading Frames Regulate PERK Translation InitiationPublication . Fernandes, Rafael; Silvestre, Samuel; Ponte, João; Lacerda, Rafaela; Romão, LuísaIntroduction: Upstream open reading frames (uORFs) are cis-acting elements located within the 5’ leader sequence (5’UTR) of transcripts, which can regulate translation of the correspondent main open reading frame (mORF). During endoplasmic reticulum (ER) stress, the accumulation of unfolded proteins activates the ER-resident PKR-like ER kinase (PERK), which results in phosphorylation of eIF2α to inhibit global mRNA translation, while allowing the selective uORF-mediated translation of downstream effectors responsible for stress resolution or, ultimately, cell death. The dual role of PERK in regulating cell fate was implicated in human diseases, like diabetes, neurodegenerative disorders and cancer. Moreover, mutations in the EIF2AK3 gene (encoding PERK) were associated to the rare genetic disease, Wolcott-Rallison Syndrome (WRS).