p53is the most known and studied tumour suppressor gene. Yet we have recently shown thatp53is als... more p53is the most known and studied tumour suppressor gene. Yet we have recently shown thatp53is also a proto-oncogene, as it encodes the Δ160p53 oncoprotein. Integrated stress response (ISR) is a survival pathway frequently activated in cancers, marked by the phosphorylation of eukaryotic initiation factor 2alpha (eIF2α) and a defined reprogramming in mRNA translation. Here we identified ISR as a powerful trigger of p53 oncogene, leading to the induction of not only Δ160p53 but also Δ133p53, another protein variant of thep53gene. Upon ISR the two isoforms were translated internally from p53 full-length (FL) transcript through an internal regulator of expression site (IRES) located in the vicinity of codon 160. Frameshift mutations upstream of codons 133 and 160 demonstrated that FLp53 protein synthesis is not required for making Δ133p53 and Δ160p53. Instead, targeting IRES(160) with an antisense oligo was sufficient to efficiently and specifically impair the expression of these isofor...
Many conditions can benefit from RNA-based therapies, namely, those targeting internal ribosome e... more Many conditions can benefit from RNA-based therapies, namely, those targeting internal ribosome entry sites (IRESs) and their regulatory proteins, the IRES trans-acting factors (ITAFs). IRES-mediated translation is an alternative mechanism of translation initiation, known for maintaining protein synthesis when canonical translation is impaired. During a stress response, it contributes to cell reprogramming and adaptation to the new environment. The relationship between IRESs and ITAFs with tumorigenesis and resistance to therapy has been studied in recent years, proposing new therapeutic targets and treatments. In addition, IRES-dependent translation initiation dysregulation is also related to neurological and cardiovascular diseases, muscular atrophies, or other syndromes. The participation of these structures in the development of such pathologies has been studied, yet to a far lesser extent than in cancer. Strategies involving the disruption of IRES–ITAF interactions or the modif...
Full-length p53 (FLp53) is a tumour suppressor protein that has been considered a master regulato... more Full-length p53 (FLp53) is a tumour suppressor protein that has been considered a master regulator of many cellular functions. Several isoforms have been described for p53 so far and some of the functions of shorter p53 isoforms have been elucidated and they are different from and complement FLp53 activity. p53 is the most commonly mutated gene in cancer and depending on its mutation status p53 may act as a tumour suppressor or a proto-oncogene. Recently, we have shown that the most common p53 cancer mutants express a larger number and higher levels of shorter p53 protein isoforms that are translated from the mutated FLp53 mRNA (Candeias et al. EMBO R. 2016). Also, we found that cells expressing these shorter p53 isoforms exhibit mutant p53 “gain-of-function” cancer phenotypes, such as enhanced cell survival, proliferation, invasion and adhesion, altered mammary tissue architecture and invasive cell structures. Here, we found that some of these mutations affect the function of an In...
In recent years, non-canonical translation initiation mechanisms have been recognized as key fact... more In recent years, non-canonical translation initiation mechanisms have been recognized as key factors in the development of different diseases such as cancer, as they present a survival answer during stress conditions by ensuring the expression of vital proteins. Internal Ribosome Entry Sites (IRESes) were first discovered on viruses, and later in eukaryotes, as mRNA secondary structures capable of recruiting the ribosome to the vicinities of an initiation codon. One of the most studied cancer-related genes, the p53 tumor suppressor gene, was found to possess on its mRNA an IRES capable of regulating the expression of the full length isoform, p53FL, and one of its isoforms, Δ40p53 differently by the interaction with MDM2 protein, an IRES trans-acting factor (ITAF) of p53. Our aim is to study a shorter p53 protein isoform that lacks tumor suppressor behaviour acting instead as a cancer promoter (Candeias et al., 2016). One of our goals is to characterize the IRES associated with its e...
The tumor microenvironment is characterized by several stresses impairing canonical translation. ... more 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, interesti...
22nd Annual Meeting of the RNA Society, 30 May -3 June 2017, May 30, 2017
The mammalian target of rapamycin (mTOR) is a conserved serine/threonine kinase that integrates s... more The mammalian target of rapamycin (mTOR) is a conserved serine/threonine kinase that integrates signals from the cellular nutrient- and energy-status, acting namely on the protein synthesis machinery. Deregulation of mTOR signaling is implicated in major diseases, such as cancer, mainly due to its role in regulating protein synthesis. Major advances are emerging regarding the regulators and effects of mTOR signaling pathway; however, regulation of mTOR gene expression is not well known. Here, we show that the 5’ untranslated region of the human mTOR transcript forms a highly folded RNA scaffold capable of binding directly to the 40S ribosomal subunit. We further demonstrate that this cis-acting RNA regulon is active both in normal and stress conditions, and that its activation status in response to translational adverse conditions parallels mTOR protein levels. Moreover, our data reveal that the cap-independent translation of mTOR is necessary for its ability to induce cell cycle progression into S-phase. These results suggest a novel regulatory mechanism of mTOR gene expression that integrates the protein profile rearrangement triggered by global translation inhibitory conditions.N/
24ª Reunião Científica da Sociedade Portuguesa de Genética Humana, 20 novembro 2020, Nov 20, 2020
Cancer is the second leading cause of death globally; therefore, its study is crucial to discover... more Cancer is the second leading cause of death globally; therefore, its study is crucial to discover new therapies. Under stress, the regular process of protein synthesis (canonical translation) is impaired, while a back-up mechanism mediated by internal ribosome entry sites (IRES) continues to function, allowing the synthesis of proteins that maintain cellular viability. This also happens in cancer cells, contributing for their survival and consequent tumorigenesis. IRES-mediated translation and its regulation by IRES trans-acting factors (ITAFs) has been correlated to metastasis and chemotherapeutic drug resistance. Therefore, our main goal was to validate ITAFs and assess their significance in cancer onset, thus becoming candidates as novel therapeutic targets. A bicistronic reporter system, which contains a first cistron translated via canonical translation and a second one translated by IRES of mTOR1 and AGO12 was used to test IRES-driven translation initiation activity. Experiments were carried out in which several proteins (hnRNPs) were silenced by specific siRNAs to analyse their function as ITAFs of mTOR and AGO1 IRESs. Also, distinct drugs were applied to simulate endoplasmic reticulum (ER) or hypoxia stress, to evaluate their effect on IRES activity. The relative IRES activity was assessed by luminescence tests and the protein levels by Western blot. In general, knockdown of hnRNPK and hnRNPU seems to decrease the IRES activity by ~60% and ~30% respectively, while hnRNPC knockdown does not show a significant effect. Regarding the ER stress, hnRNPK knockdown seems to decrease even more the IRES activity, while hnRNPU depletion induces a significant increase. On the other hand, in hypoxia, the hnRNPs knockdowns do not significantly affect IRES activity. These results indicate that hnRNPK and hnRNPU may function as ITAFs of mTOR and AGO1 IRES activity in cells under ER stress. Our data can be decisive for a better understanding of carcinogenesis and suggest new therapeutic targets for cancer treatment. 1. Marques-Ramos, A., et.al. 2017. RNA. 23, 1712-1728 2. Lacerda, R. 2016. Faculdade de Ciências e Tecnologia da Universidade NOVA de LisboaWork partially supported by UID/MULTI/04046/2019 Research Unit grant from FCT, Portugal (to BioISI)info:eu-repo/semantics/publishedVersio
This work is financed by Fundacao para a Ciencia e a Tecnologia (PTDC/BIMONC/4890/2014 to MMC and... more This work is financed by Fundacao para a Ciencia e a Tecnologia (PTDC/BIMONC/4890/2014 to MMC and UID/MULTI/04046/2013 to BioISI from FCT/MCTES/PIDDAC.
This work is partially supported by Fundacao para a Ciencia e a Tecnologia (PTDC/BIMONC/4890/2014... more This work is partially supported by Fundacao para a Ciencia e a Tecnologia (PTDC/BIMONC/4890/2014), by Grants-in-Aid 16K21111 and 18K07229 from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan and by Takeda Foundation.
This work was partially supported by Fundacao Merck Sharp and Dohme and Fundacao para a Ciencia e... more This work was partially supported by Fundacao Merck Sharp and Dohme and Fundacao para a Ciencia e a Tecnologia (FCT) through center grant UID/MULTI/04046/2013 (to BioISI) and research grant PTDC/BIM-ONC/4890/2014. Ana Marques-Ramos, Juliane Menezes, and Rafaela Lacerda were supported by Fellowships from FCT [SFRH/BD/33462/2008 to A.M.-R., FCT/SFRH/BPD/98360/2013 to J.M., and SFRH/BD/74778/2010 to R.L.]. Marco Marques Candeias was supported by fellowships from the Japan Society for the Promotion of Science (JSPS/FF1/184) and AXA Research Fund and JSPS Grant-in-Aid for Young Scientists (B) (16K21111).
p53is the most known and studied tumour suppressor gene. Yet we have recently shown thatp53is als... more p53is the most known and studied tumour suppressor gene. Yet we have recently shown thatp53is also a proto-oncogene, as it encodes the Δ160p53 oncoprotein. Integrated stress response (ISR) is a survival pathway frequently activated in cancers, marked by the phosphorylation of eukaryotic initiation factor 2alpha (eIF2α) and a defined reprogramming in mRNA translation. Here we identified ISR as a powerful trigger of p53 oncogene, leading to the induction of not only Δ160p53 but also Δ133p53, another protein variant of thep53gene. Upon ISR the two isoforms were translated internally from p53 full-length (FL) transcript through an internal regulator of expression site (IRES) located in the vicinity of codon 160. Frameshift mutations upstream of codons 133 and 160 demonstrated that FLp53 protein synthesis is not required for making Δ133p53 and Δ160p53. Instead, targeting IRES(160) with an antisense oligo was sufficient to efficiently and specifically impair the expression of these isofor...
Many conditions can benefit from RNA-based therapies, namely, those targeting internal ribosome e... more Many conditions can benefit from RNA-based therapies, namely, those targeting internal ribosome entry sites (IRESs) and their regulatory proteins, the IRES trans-acting factors (ITAFs). IRES-mediated translation is an alternative mechanism of translation initiation, known for maintaining protein synthesis when canonical translation is impaired. During a stress response, it contributes to cell reprogramming and adaptation to the new environment. The relationship between IRESs and ITAFs with tumorigenesis and resistance to therapy has been studied in recent years, proposing new therapeutic targets and treatments. In addition, IRES-dependent translation initiation dysregulation is also related to neurological and cardiovascular diseases, muscular atrophies, or other syndromes. The participation of these structures in the development of such pathologies has been studied, yet to a far lesser extent than in cancer. Strategies involving the disruption of IRES–ITAF interactions or the modif...
Full-length p53 (FLp53) is a tumour suppressor protein that has been considered a master regulato... more Full-length p53 (FLp53) is a tumour suppressor protein that has been considered a master regulator of many cellular functions. Several isoforms have been described for p53 so far and some of the functions of shorter p53 isoforms have been elucidated and they are different from and complement FLp53 activity. p53 is the most commonly mutated gene in cancer and depending on its mutation status p53 may act as a tumour suppressor or a proto-oncogene. Recently, we have shown that the most common p53 cancer mutants express a larger number and higher levels of shorter p53 protein isoforms that are translated from the mutated FLp53 mRNA (Candeias et al. EMBO R. 2016). Also, we found that cells expressing these shorter p53 isoforms exhibit mutant p53 “gain-of-function” cancer phenotypes, such as enhanced cell survival, proliferation, invasion and adhesion, altered mammary tissue architecture and invasive cell structures. Here, we found that some of these mutations affect the function of an In...
In recent years, non-canonical translation initiation mechanisms have been recognized as key fact... more In recent years, non-canonical translation initiation mechanisms have been recognized as key factors in the development of different diseases such as cancer, as they present a survival answer during stress conditions by ensuring the expression of vital proteins. Internal Ribosome Entry Sites (IRESes) were first discovered on viruses, and later in eukaryotes, as mRNA secondary structures capable of recruiting the ribosome to the vicinities of an initiation codon. One of the most studied cancer-related genes, the p53 tumor suppressor gene, was found to possess on its mRNA an IRES capable of regulating the expression of the full length isoform, p53FL, and one of its isoforms, Δ40p53 differently by the interaction with MDM2 protein, an IRES trans-acting factor (ITAF) of p53. Our aim is to study a shorter p53 protein isoform that lacks tumor suppressor behaviour acting instead as a cancer promoter (Candeias et al., 2016). One of our goals is to characterize the IRES associated with its e...
The tumor microenvironment is characterized by several stresses impairing canonical translation. ... more 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, interesti...
22nd Annual Meeting of the RNA Society, 30 May -3 June 2017, May 30, 2017
The mammalian target of rapamycin (mTOR) is a conserved serine/threonine kinase that integrates s... more The mammalian target of rapamycin (mTOR) is a conserved serine/threonine kinase that integrates signals from the cellular nutrient- and energy-status, acting namely on the protein synthesis machinery. Deregulation of mTOR signaling is implicated in major diseases, such as cancer, mainly due to its role in regulating protein synthesis. Major advances are emerging regarding the regulators and effects of mTOR signaling pathway; however, regulation of mTOR gene expression is not well known. Here, we show that the 5’ untranslated region of the human mTOR transcript forms a highly folded RNA scaffold capable of binding directly to the 40S ribosomal subunit. We further demonstrate that this cis-acting RNA regulon is active both in normal and stress conditions, and that its activation status in response to translational adverse conditions parallels mTOR protein levels. Moreover, our data reveal that the cap-independent translation of mTOR is necessary for its ability to induce cell cycle progression into S-phase. These results suggest a novel regulatory mechanism of mTOR gene expression that integrates the protein profile rearrangement triggered by global translation inhibitory conditions.N/
24ª Reunião Científica da Sociedade Portuguesa de Genética Humana, 20 novembro 2020, Nov 20, 2020
Cancer is the second leading cause of death globally; therefore, its study is crucial to discover... more Cancer is the second leading cause of death globally; therefore, its study is crucial to discover new therapies. Under stress, the regular process of protein synthesis (canonical translation) is impaired, while a back-up mechanism mediated by internal ribosome entry sites (IRES) continues to function, allowing the synthesis of proteins that maintain cellular viability. This also happens in cancer cells, contributing for their survival and consequent tumorigenesis. IRES-mediated translation and its regulation by IRES trans-acting factors (ITAFs) has been correlated to metastasis and chemotherapeutic drug resistance. Therefore, our main goal was to validate ITAFs and assess their significance in cancer onset, thus becoming candidates as novel therapeutic targets. A bicistronic reporter system, which contains a first cistron translated via canonical translation and a second one translated by IRES of mTOR1 and AGO12 was used to test IRES-driven translation initiation activity. Experiments were carried out in which several proteins (hnRNPs) were silenced by specific siRNAs to analyse their function as ITAFs of mTOR and AGO1 IRESs. Also, distinct drugs were applied to simulate endoplasmic reticulum (ER) or hypoxia stress, to evaluate their effect on IRES activity. The relative IRES activity was assessed by luminescence tests and the protein levels by Western blot. In general, knockdown of hnRNPK and hnRNPU seems to decrease the IRES activity by ~60% and ~30% respectively, while hnRNPC knockdown does not show a significant effect. Regarding the ER stress, hnRNPK knockdown seems to decrease even more the IRES activity, while hnRNPU depletion induces a significant increase. On the other hand, in hypoxia, the hnRNPs knockdowns do not significantly affect IRES activity. These results indicate that hnRNPK and hnRNPU may function as ITAFs of mTOR and AGO1 IRES activity in cells under ER stress. Our data can be decisive for a better understanding of carcinogenesis and suggest new therapeutic targets for cancer treatment. 1. Marques-Ramos, A., et.al. 2017. RNA. 23, 1712-1728 2. Lacerda, R. 2016. Faculdade de Ciências e Tecnologia da Universidade NOVA de LisboaWork partially supported by UID/MULTI/04046/2019 Research Unit grant from FCT, Portugal (to BioISI)info:eu-repo/semantics/publishedVersio
This work is financed by Fundacao para a Ciencia e a Tecnologia (PTDC/BIMONC/4890/2014 to MMC and... more This work is financed by Fundacao para a Ciencia e a Tecnologia (PTDC/BIMONC/4890/2014 to MMC and UID/MULTI/04046/2013 to BioISI from FCT/MCTES/PIDDAC.
This work is partially supported by Fundacao para a Ciencia e a Tecnologia (PTDC/BIMONC/4890/2014... more This work is partially supported by Fundacao para a Ciencia e a Tecnologia (PTDC/BIMONC/4890/2014), by Grants-in-Aid 16K21111 and 18K07229 from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan and by Takeda Foundation.
This work was partially supported by Fundacao Merck Sharp and Dohme and Fundacao para a Ciencia e... more This work was partially supported by Fundacao Merck Sharp and Dohme and Fundacao para a Ciencia e a Tecnologia (FCT) through center grant UID/MULTI/04046/2013 (to BioISI) and research grant PTDC/BIM-ONC/4890/2014. Ana Marques-Ramos, Juliane Menezes, and Rafaela Lacerda were supported by Fellowships from FCT [SFRH/BD/33462/2008 to A.M.-R., FCT/SFRH/BPD/98360/2013 to J.M., and SFRH/BD/74778/2010 to R.L.]. Marco Marques Candeias was supported by fellowships from the Japan Society for the Promotion of Science (JSPS/FF1/184) and AXA Research Fund and JSPS Grant-in-Aid for Young Scientists (B) (16K21111).
Uploads
Papers by Rafaela Lacerda