Browsing by Author "Candeias, Marco M."
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- Alternative mechanisms of mRNA translationiInitiation in cellular stress response and cancerPublication . Lacerda, Rafaela; Menezes, Juliane; Candeias, Marco M.Throughout evolution, eukaryotic cells have devised different mechanisms to cope with stressful environments. When eukaryotic cells are exposed to stress stimuli, they activate adaptive pathways that allow them to restore cellular homeostasis. Most types of stress stimuli have been reported to induce a decrease in overall protein synthesis accompanied by induction of alternative mechanisms of mRNA translation initiation. Here, we present well-studied and recent examples of such stress responses and the alternative translation initiation mechanisms they induce, and discuss the consequences of such regulation for cell homeostasis and oncogenic transformation.
- Characterization of an oncogenic isoform of TP53: Δ160p53Publication . Ramalho, Ana Catarina; Rita, Filipa; López-Iniesta, M.; Lacerda, Rafaela; Romão, Luísa; Candeias, Marco M.The transcription factor p53 is a key cell regulator, having roles in varied cellular processes. Widely known as a tumour suppressor protein, p53 is responsible for signalling the adequate response to DNA damage, oncogenic signalling, or other stress stimuli. The target genes of this protein are involved in cell cycle arrest, senescence, apoptosis, and DNA damage response, among other pathways. Besides the full-length p53 (FLp53), to which these functions are attributed, the TP53 gene encodes for eleven other protein isoforms that result from alternative splicing, internal initiation of translation and transcription from an internal promoter. In striking contrast to FLp53, the N-terminally truncated Δ160p53 exhibits pro-oncogenic traits, although it only differs from FLp53 by the lack of its first 159 amino acids. Δ160p53 promotes cell survival, proliferation, invasion, and adhesion, and it is overexpressed in cancer cells harbouring hotspot p53 mutants. The TP53 gene is frequently mutated in cancer, and hotspot mutants result from single missense mutations that convert p53 in a driver of tumorigenesis. As Δ160p53 presents many of the oncogenic roles attributed to p53 cancer mutants, it is plausible that this isoform could be responsible for the paradoxical mutant p53 functions. However, detailed knowledge on the mode of action of Δ160p53 is still lacking. We have performed additional tests to further characterize the oncogenic traits of this isoform. To evaluate the ability of Δ160p53 to promote anchorage-independent cell growth, we have used the soft agar colony formation assay. Preliminary results show a tendency of Δ160p53 to promote growth, when compared to the control and other isoforms. Our new data complements our previous knowledge on Δ160p53 and reinforces the importance of studying this isoform for therapeutic targeting.
- Human TP53 is a tumour suppressor that acquires oncogenic functions during integrated stress response (ISR) due to a translational switchPublication . Lacerda, Rafaela; Fonseca Costa, Inês; López-Iniesta, Maria; Romão, Luísa; Candeias, Marco M.Eukaryotic cells have developed different mechanisms and adaptive pathways that allow them to cope with external stress stimuli. Under stress conditions, global protein synthesis is shut down, and some alternative mechanisms of mRNA translation initiation are induced. Although the tumour suppressor protein p53 — the most mutated gene in cancer — has been considered the guardian of the genome and a master regulator of several cellular functions, the truth is it is not just one isoform, the full-length (FLp53), but also many other p53 isoforms that have been described so far. Based on our previous results, some functions of the shorter isoforms are different from and complement FLp53 activity. Here we show the specific induction of Δ160p53 isoform during integrated stress response (ISR). We confirmed the presence of an Internal Ribosome Entry Site (IRES) in p53 mRNA that controls Δ160p53 isoform translation, using a bicistronic reporter construct. When subjecting cells to endoplasmic reticulum stress, we showed that eIF2α phosphorylation is a key event leading to cap-independent expression of Δ160p53 during ISR. Also, some cancer-specific mutations in the DNA-binding domain of p53 enhance cap-independent translation of Δ160p53 via Δ160p53IRES. Using an antisense morpholino oligo targeting Δ160IRES significantly reduces Δ160p53 protein levels and impaired its oncogenic functions. Additionally, we found the 5’untranslated region of Δ160p53 inhibits the IRES activity. Our data support a model in which an IRES structure in the coding region of p53 is activated under stress conditions, leading to the expression of the oncogenic shorter Δ160p53 isoform, whose structure is affected by cancer-specific mutations in the p53 gene. A better understanding of Δ160p53IRES structure and function may be advantageous for a more efficient therapeutic targeting of p53.
- Regulation of IRES-mediated translation in p53Publication . Fonseca Costa, Inês; Lacerda, Rafaela; López-Iniesta, M.; Romão, Luísa; Candeias, Marco M.The tumor microenvironment is characterized by several stresses impairing canonical translation. However, specific mRNAs harbouring internal ribosome entry sites (IRES), such as several tumour suppressors and oncogenes, can overcome this impairment. The tumor suppressor TP53 gene, an important transcription factor that ensures cellular homeostasis, is frequently mutated in human cancers. Over the years, several p53 isoforms have been identified, which in some cases result from alternative initiation of translation regulated by an IRES. Recently, we have associated mutant p53 “gain-of-function” cancer phenotype, such as enhanced cell survival, invasion, proliferation, and adhesion, with the expression of higher levels of shorter p53 isoforms, such as Δ160p53 isoform.1 Here, we used a bicistronic system containing two reporter luciferases (renilla luciferase and firefly luciferase) to assess IRES-mediated translation. Several p53 mRNA elements were tested in this system and, interestingly, we have found an inhibitory element of IRES-mediated translation. Overall, IRES-regulated translation in malignant cells is used to translate specific proteins that promote cancer progression. Thus, inhibiting translation of oncogenes via IRES could prevent the formation of tumor cells and their adaptation to unfavourable conditions in the tumor microenvironment. 1. Candeias, M. M., Hagiwara, M. & Matsuda, M. Cancer‐specific mutations in p53 induce the translation of Δ160p53 promoting tumorigenesis. EMBO Rep. 17, 1542–1551 (2016).
- RNA structure-function analysis of regulatory regions of p53 mRNAPublication . Pereira, Bruna F.; López-Iniesta, Maria; Lacerda, Rafaela; Romão, Luísa; Candeias, Marco M.At 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.
- The integrated stress response releases the oncoprotein in TP53Publication . Ramalho, Ana Catarina; López-Iniesta, Maria José; Lacerda, Rafaela; Parker, Shrutee N.; Romão, Luísa; Candeias, Marco M.Introduction: The TP53 gene is surrounded by a great duality: it is a critical tumour suppressor gene, however, its protective nature is frequently lost in tumours, where it becomes a powerful oncogene. Numerous studies attribute the oncogenic profile of TP53 to the missense mutations that commonly occur in cancer. We propose that this duality is intrinsic to TP53 instead of a consequence of mere somatic mutations, which could not have been evolutionarily selected for. This gene encodes for a set of protein isoforms with distinct and complementary functions, from which the full-length p53 (FLp53) is the best characterized. FLp53 is a transcription factor that mediates stress responses by promoting cell cycle arrest, DNA repair or apoptosis. In stark contrast to FLp53, the shorter isoform Δ160p53 promotes cell survival, proliferation, and invasion, and it is commonly overexpressed in tumours. Here we identify a disruption in the normal balance of p53 isoforms upon induction of the Integrated Stress Response (ISR), with the translation of Δ160p53 being favoured. Materials and Methods: Different cell lines were used to verify the expression of endogenous p53 isoforms during ISR, and the internal translation of Δ160p53 was tested with bicistronic constructs. The interaction of Δ160p53 with FLp53 was assessed by co-immunoprecipitation followed by western blot, and its effect in the expression of target genes was measured by RT-qPCR. Cells were treated with thapsigargin and tunicamycin to induce ISR when required. Results: The induction of ISR in a group of cell lines led to increased levels of endogenous Δ160p53 protein, as well as increased luminescence signal in the bicistronic system. The FLp53-Δ160 interaction was confirmed, and the role of Δ160p53 in the selective regulation of p53 target genes was uncovered. Conclusions: These data uncover a mode of activation of the oncogenic Δ160p53 and how this isoform can work together with FLp53. In the future, we aim to explore the clinical potential of these discoveries.
- The p53 short isoforms are activated by the integrated stress response to enhance survival in wild-type p53 cancer cell linesPublication . Ramalho, Ana Catarina; López-Iniesta, Maria José; Lacerda, Rafaela; Parker, Shrutee N.; Romão, Luísa; Candeias, Marco M.Introduction: The full-length p53 (FLp53) protein is a transcriptional factor that mediates cellular stress responses. The appropriate response is modulated according to the nature and extent of the damage, which means that p53 may promote cell cycle arrest and DNA repair or apoptosis in different situations. In all cases, p53 manages the response by modulating the expression of a wide range of target genes, as it contains two N-terminal transactivation domains. However, this gene also encodes for shorter isoforms that lack the N-terminal region and display oncogenic functions: Δ133p53 and Δ160p53. In contrast to the protective nature of the full-length protein, these two isoforms promote cell survival, proliferation and invasion. Both are typically overexpressed in tumours, while in normal tissues their levels are low or undetectable. Data from this study indicate that the elevated levels of these isoforms may be owed to the Integrated Stress Response (ISR), which is typically a pro-survival programme, and it is commonly activated in cancer cells. The ISR is initiated by a group of kinases in response to different stress stimuli, converging in the phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α). This blocks global translation, while promoting the expression of activating transcription factor 4 (ATF4), the main effector of ISR. Materials and Methods: In this work, cancer cell lines with endogenous p53 were used to verify the expression of its isoforms during ISR, which was induced by treatment with thapsigargin and tunicamycin. To investigate if they were translated internally, bicistronic construct systems were employed. The interaction of the isoforms with FLp53 was verified by co-immunoprecipitation and their effect on the mRNA levels of p53 target genes was evaluated. Results and discussions: The translation of Δ160p53 and Δ133p53 was promoted by ISR induction in the cell lines tested via internal translation from FLp53 mRNA. Interaction of both isoforms with FLp53 was also confirmed, and it was demonstrated that Δ160p53 selectively acts as a regulator of p53 transcriptional activity on some genes. Conclusion: These results hint at a physiological role for p53 short isoforms in the modulation of p53 target gene expression during ISR. On the other hand, their activation by abnormal ISR induction in cancer cells supports oncogenicity. The uncovering of an intersection between p53 isoforms and ISR could open a new path for future cancer therapies.
- The tumor suppressor p53 acquires oncogenic functions due to a translational switch during integrated stress responsePublication . Lacerda, Rafaela; Fonseca Costa, Inês; López-Iniesta, M.; Romão, Luísa; Candeias, Marco M.To cope with the stress stimuli to which they are often exposed, eukaryotic cells have developed adaptive pathways that restore cellular homeostasis. Under stress conditions there is an overall decrease of protein synthesis, and a concomitant induction of alternative mechanisms of mRNA translation initiation. The tumour suppressor protein p53 has been considered the guardian of the genome and a master regulator of many cellular functions. However, apart from the full-length p53 (FLp53), several p53 isoforms have been described so far. Some functions of shorter p53 isoforms have already been elucidated and they are different from and complement FLp53 activity, the most mutated gene in cancer. Here we show that the integrated stress response (ISR) leads to the specific induction of Δ160p53 isoform. Using bicistronic constructs we confirmed the presence of an Internal Ribosome Entry Site (IRES) in p53 mRNA that controls Δ160p53 isoform translation. Subjecting cells to endoplasmic reticulum stress showed that eIF2α phosphorylation is a key event leading to cap-independent expression of Δ160p53 during ISR. Additionally, cancer-specific mutations in p53 also enhanced cap-independent translation of Δ160p53 via Δ160p53IRES. An antisense morpholino oligo targeting Δ160IRES efficiently reduced Δ160p53 protein levels and significantly impaired oncogenic functions of Δ160p53. Our data support a model in which an IRES structure in the coding region of p53 is activated under stress conditions, leading to the expression of the oncogenic shorter Δ160p53 isoform, whose structure is affected by cancer-specific mutations in the p53 gene. A better understanding of Δ160p53IRES structure and function may be advantageous for a more efficient therapeutic targeting of p53.
- Translational Control of Δ160p53 Keeps the Dark Side of TP53 in CheckPublication . Ramalho, Ana Catarina; Lacerda, Rafaela; Romão, Luísa; Candeias, Marco M.The TP53 tumour suppressor gene was discovered over 40 years ago, but to this day some aspects of its regulation and function remain a mystery. It encodes the full-length p53 protein (FLp53), a transcription factor with a key role in stress response in multicellular organisms, that can either direct cells towards apoptosis or recovery of homeostasis. With such a decisive role, its expression and activity are tightly regulated. A vast set of RNA-binding proteins (RBPs) have been described to affect the translation of FLp53 or the stability of p53 mRNA in response to different perturbations. But in addition to FLp53, there is a group of shorter protein isoforms lacking the N-terminal region, which have well-described functions, and are translated from the same mRNA. The shorter and less studied isoform is Δ160p53, which promotes cell survival, proliferation, and invasion. Despite its usual low levels, it is commonly overexpressed in tumours. However, the detailed mechanisms and factors involved in the regulation of Δ160p53 are still unknown. In this work, a mass spectrometry was performed to identify the proteins in an RNA-protein co-immunoprecipitation of the p53 mRNA using the MS2 system in the p53-null cell line H1299. The validation of the hits was undertaken by western blot with specific antibodies after immunoprecipitation. The effect of the binding proteins on the translation of Δ160p53 was assessed by overexpression or knockdown, and the expression levels were verified by western blot or luminescence assays. The mass spectrometry allowed the identification of potential new binding partners of the p53 mRNA. Resorting to the literature and to computational tools available online to predict protein-RNA interactions, a few hits were selected for follow-up and their interactions confirmed. Simultaneously, the modulation of Δ160p53 expression by some of these proteins was verified. Considering the importance of TP53 in deciding the fate of the cell, the observation of abnormal levels of the oncoprotein Δ160p53 in cancer is intriguing. Understanding the control of its translation could uncover strategies to block it and pave way for new cancer therapies.