Browsing by Author "Pereira, Bruna"
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- Common p53 mutations induce IRES-mediated translation of oncogenic shorter p53 isoformsPublication . Pereira, Bruna; Lacerda, Rafaela; Maria López-Iniesta, M; Romão, Luísa; Candeias, MarcoAt least half of all tumors exhibit mutations in the tumor suppressor p53 gene. Indeed, the fact that p53 is frequently mutated in cancer led to its identification as an oncogene, when first described in 1979. Later, it was classified as a tumor suppressor, due to the clarification of its wild-type role in maintaining genome integrity and preventing malignant transformation. The p53 gene can encode for many p53 isoforms, by alternative splicing, alternative promoters and internal translation initiation mechanisms. While full-length p53 (FL-p53) protein works as a tumor suppressor by regulating many biological processes such as cell cycle, apoptosis, senescence and DNA repair, shorter p53 protein isoforms seem to play different roles in the cell. Recently, we have shown that the most common p53 mutations induce the expression of shorter p53 isoforms. Furthermore, we found that shorter p53 isoforms are implicated in cancer progression as they promote enhanced cell survival, proliferation, adhesion and formation of invasive cell structures. Here, with a bicistronic system containing two reporter genes (Renilla luciferase and firefly luciferase), we show that expression of shorter p53 isoforms is mediated by a non-canonical translation initiation mechanism regulated by an Internal Ribosome Entry Site (IRES) in the p53 mRNA. By investigating the effect of common p53 missense mutations on the function of this new IRES, through bioluminescence assays and Western blot analysis, we show that some p53 cancer mutations have a preponderant role in IRES-mediated translation induction of shorter p53 isoforms. With the obtained results we identified a new mechanism by which p53 cancer mutations promote tumorigenesis, which may lead to new understandings of the onset and progression of some types of tumors as well as to the development of new cancer therapies.
- Genetics of personalized medicine: cancer and rare diseasesPublication . Siefers Alves, Inês; Condinho, Manuel; Custódio, Sónia; Pereira, Bruna; Fernandes, Rafael; Gonçalves, Vânia; da Costa, Paulo J.; Lacerda, Rafaela; Marques, Ana Rita; Martins-Dias, Patrícia; Nogueira, Gonçalo; Neves, Ana Rita; Pinho, Patrícia; Rodrigues, Raquel; Rolo, Eva; Silva, Joana; Travessa, André; Pinto-Leite, Rosário; Sousa, Ana; Romão, LuísaThe 21st annual meeting of the Portuguese Society of Human Genetics (SPGH), organized by Luísa Romão, Ana Sousa and Rosário Pinto Leite, was held in Caparica, Portugal, from the 16th to the 18th of November 2017. Having entered an era in which personalized medicine is emerging as a paradigm for disease diagnosis, treatment and prevention, the program of this meeting intended to include lectures by leading national and international scientists presenting exceptional findings on the genetics of personalized medicine. Various topics were discussed, including cancer genetics, transcriptome dynamics and novel therapeutics for cancers and rare disorders that are designed to specifically target molecular alterations in individual patients. Several panel discussions were held to emphasize (ethical) issues associated with personalized medicine, including genetic cancer counseling.
- Study on the regulation of the expression of alternative protein isoforms involved in carcinogenesisPublication . Pereira, Bruna; Loison, Luísa Romão; Candeias, Marco MarquesIn eukaryotes, gene expression is a highly complex process composed of several steps. One of those is translation, the step that converts the genetic information contained in the messenger ribonucleic acid (mRNA) into functional proteins. Under normal conditions, most proteins are translated through the canonical translation initiation mechanism, which starts with cap structure recognition at the 5’-end of mRNAs, followed by 5’ UTR (untranslated region) scanning until the appearance of an initiation codon in a favorable context. Yet, under unfavorable or energy-depriving conditions, such as endoplasmic reticulum (ER) stress, hypoxia, nutrient starvation, mitosis and cell differentiation, canonical translation is impaired and protein synthesis globally decreases. Nevertheless, some mRNAs, usually related to stress-responses, cell growth and cell death control, continue to be translated through alternative mechanisms. One of them involves internal ribosome entry sites (IRES), in which the ribosome is directly recruited to the vicinity of the initiation codon, without requiring the cap structure. This mechanism relies on mRNA secondary structures and can be assisted by some canonical factors and other auxiliary proteins named ITAFs (IRES trans-acting factors). Tumor cells take advantage of this mechanism to cope with the unfavorable conditions that characterize tumor microenvironment and proliferate. Indeed, many mRNAs containing IRES elements are found deregulated in cancer. One example is the tumor suppressor p53. The TP53 gene is the most commonly mutated gene in cancer and surprisingly, p53 mutations usually lead to the production of a mutant protein with oncogenic functions. The presence of three promoters on TP53 gene leads to the expression of different transcripts expressing different alternative translation products: the full-length transcripts allow the expression of FL-p53, Δ40p53 and Δ160p53, and a shorter transcript produces Δ133p53 and also Δ160p53. While FL-p53 and Δ40p53 protein isoforms have been widely studied in terms of internal initiation mechanisms, the fact that Δ160p53 expression is mediated through an IRES element was not known until recently. Since this shorter p53 isoform was already associated with survival, proliferation and invasion of tumor cells, the recently identified Δ160p53 IRES may have an important role in tumorigenic functions of Δ160p53. Thus, considering the aforementioned data, we proposed to study the regulation of the expression of alternative protein isoforms involved in carcinogenesis, more specifically, the regulation of Δ160p53 expression through its IRES element, aiming to understand the role of IRES-mediated translation in cancer development. Knowing that Δ160p53 IRES is located within the first 432 nucleotides of Δ160p53 coding sequence and that its activity is inhibited by Δ160p53 5’ UTR, we evaluated the effect of hotspot p53 missense mutations (R175H, R248Q, R273H e R282W) in reverting the inhibitory effect of Δ160p53 5’ UTR on Δ160p53 IRES activity. To do that, we used a bicistronic system containing two reporter genes: Renilla Luciferase (RLuc) and firefly Luciferase (FLuc). RLuc is the first cistron and its expression is driven by cap-dependent mechanisms, while FLuc is the second cistron and is only translated if there is an upstream sequence capable of promoting its translation through cap-independent mechanisms. In our experiments, we were able to see the inhibitory effect of Δ160p53 5’ UTR on Δ160p53 IRES activity, in HeLa cells, corroborating the previous reported results. Moreover, from all tested p53 missense mutations (R175H, R248Q, R273H e R282W), only R175H was capable of reverting some of the 5’ UTR inhibitory effect on Δ160p53 IRES activity. This was observed for cells under 2 μM Thapsigargin-induced ER stress, which is known to impair cap-dependent translation. The obtained results seem to indicate that R175H oncogenic functions go beyond the alteration or loss of protein function, since this mutation also appears to act through an mRNA-dependent manner by inducing the expression of Δ160p53, an isoform that has already been shown to have importance in promoting tumorigenesis. Furthermore, in this thesis, we also aimed the identification of Δ160p53 IRES auxiliary proteins, using a system that takes advantage of MS2 RNA–MS2 coat protein interaction. Cloning p53 sequences of interest, such as the Δ160p53 IRES, upstream of MS2 RNA repeats, followed by co-transfection of these constructs with that expressing the MS2 coat protein and co-immunoprecipitation, will allow the identification of p53 mRNA-interacting proteins, through mass spectrometry. Here, we describe some of the cloning strategies used, though unsuccessfully, to attempt to clone p53 sequences of interest upstream MS2 repeats as well assome possible solutions. Moreover, knowing that Hdm2 (murine double minute 2 human homolog) interacts with several mRNAs commonly deregulated in cancer cells, such as p53 and XIAP (X-linked inhibitor of apoptosis protein), regulating their non-canonical translation, we also performed Hdm2 immunoprecipitation optimizations, so that, in the future, co-immunoprecipitation of Hdm2-bound mRNAs can be performed. Then, new possible IRES-containing mRNAs regulated by Hdm2 that may also have a preponderant role in cancer progression will be identified by RNA sequencing. At the end, concluding all these lines of research, we hope to unveil new insights regarding IRES-mediated translation of cancer-related mRNAs. In fact, understanding how IRES-containing mRNAs are regulated under different stress conditions and how the switch between cell homeostasis and cell neoplastic transformation is triggered, will provide important knowledge for the development of new therapeutic strategies.
- Translational switch during integrated stress response: the examples of p53 and UPF1Publication . Lacerda, Rafaela; Pereira, Bruna; Menezes, Juliane; Ramos, Ana; Neves, Ana Rita; Candeias, Marco M; Romão, LuísaThe scanning model for eukaryotic mRNA translation initiation states that the small ribosomal subunit, along with initiation factors, binds to the cap structure at the 5’ end of the mRNA and scans the 5’ untranslated region (5’UTR) until an initiation codon is found. However, when cells are exposed to stress stimuli, cap-dependent translation is inhibited, while the synthesis of some proteins is maintained by alternative mechanisms of translation initiation, which are vital for cell survival and stress recovery. Here we show two examples in which a translational switch occurs during integrated stress response (ISR). In the first case, tumor suppressor p53, we show that the ISR leads to the specific induction of a shorter p53 isoform (Δ160p53 isoform). This induction is dependent on translation elongation but does not require the eIF4E-eIF4G interaction. Studies using bicistronic constructs with wild-type Δ160p53 or reporter genes confirmed the presence of an Internal Ribosome Entry Site (IRES) in p53 mRNA, being eIF2α phosphorylation a key event leading to cap-independent expression of Δ160p53 during ISR. Interestingly, cancer-specific mutations in p53 also enhance cap-independent translation of Δ160p53 via Δ160p53IRES. Our data support a model in which an IRES structure in the coding region of p53, and the cancer-specific mutations that affect this structure, control p53 oncogenic functions by regulating Δ160p53 protein expression. A better understanding of Δ160p53IRES structure and function may be advantageous for a more efficient therapeutic targeting of p53. Human up-frameshift 1 (UPF1) is a key-protein involved in nonsense-mediated mRNA decay, telomere replication and homeostasis, and cell cycle progression. These crucial UPF1 functions suggest its tight gene expression regulation. Indeed, our results show that UPF1 5’UTR is able to mediate cap-independent translation in a bicistronic luciferase vector expressed in cervical and colorectal cancer cell lines. Such activity is maintained under endoplasmic reticulum stress. Interestingly, we found that the UPF1 5’UTR IRES function is inhibited when the first 100 nucleotides, or the last 125, are absent or altered. Understanding these IRESs mechanism of function and their biological relevance might provide tools for developing new therapies for human diseases such as cancer.