Unraveling the oncogenic functions and regulation of the most conserved p53 isoform, 160p53: a study in cancer cells and transgenic mice

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Conserved Double Translation Initiation Site for Δ160p53 Protein Hints at Isoform's Key Role in Mammalian Physiology

Publication . López-Iniesta, Maria José; Parker, Shrutee; Ramalho, Ana Catarina; Lacerda, Rafaela; Costa, Inês; Zhao, Jingyuan; Romão, Luísa; Candeias, Marco

p53 is the most commonly mutated gene in human cancers. Two fundamental reasons for this are its long protein isoforms protect from cancer, while its shorter C-terminal isoforms can support cancer and metastasis. Previously, we have shown that the Δ160p53 protein isoform enhances survival and the invasive character of cancer cells. Here, we identified a translation initiation site nine codons downstream of codon 160-the known initiation codon for the translation of Δ160p53-that is recognized by the translation machinery. When translation failed to initiate from AUG160 due to mutation, it initiated from AUG169 instead, producing similar levels of a similar protein, Δ169p53, which promoted cell survival as efficiently as Δ160p53 following DNA damage. Interestingly, almost all mammalian species with an orthologue to human AUG160 also possess one for AUG169, while none of the non-mammalian species lacking AUG160 have AUG169, even if that region of the p53 gene is well conserved. In view of our findings, we do not believe that Δ169p53 acts as a different p53 protein isoform; instead, we propose that the double translation initiation site strengthens the translation of these products with a critical role in cell homeostasis. Future studies will help verify if this is a more general mechanism for the expression of essential proteins in mammals.

2022-12-13Journal article

Open access

The p53 short isoforms are activated by the integrated stress response to enhance survival in wild-type p53 cancer cell lines

Publication . 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.

2023-06-12Conference object

Restricted access

The integrated stress response releases the oncoprotein in TP53

Publication . 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.

2023-10-26Conference object

Open access