Given its role as “guardian of the genome”, the protein p53 has been extensively studied in the cancer research field, with the aim of exploiting its potential in cancer therapies. Nutlin-3 can be considered the prototype molecule able to activate WT p53 by inhibiting the interaction with its negative regulator MDM2. Previous evidence coming from literature and experiments performed by other members of the laboratory showed how cell lines undergo different responses when treated with Nutlin-3. Specifically, colon cancer-derived HCT116 cells undergo cell cycle arrest when treated with 10µM of Nutlin-3a for 48 hours, while SJSA1 (osteosarcoma-derived) undergo massive apoptosis. Transcription regulation alone cannot account for the antithetic behaviour of the cell lines since apoptotic factors are transcriptionally modulated in both. Conversely, the analysis of polysome-bound mRNAs revealed a core of differentially regulated genes that share a CG-motif in the 3’UTR whose expression is enhanced in Nutlin-3-treated SJSA1 cells. A luciferase-based assay confirmed that the addition of this motif to a 3’UTR is sufficient to enhance translation of the reporter gene in SJSA1 but not in HCT116 cells treated with Nutlin-3. Analysis of the interactors of the CG-motif by pulldown followed by mass spectrometry revealed a core of interacting RBPs shared between the two cell lines and two specific interactors, DHX30 and MYH9. On the other hand, since the motif was showing a biased composition towards cytosines, we analysed the expression of PCBP family proteins, revealing a differential expression of PCBP2 between HCT116 and SJSA1. The role of PCBP2 and DHX30 was tested in HCT116 cells, being the cell line expressing higher levels of both RBPs. We confirmed the ability of these proteins to interact with CG-motif containing RNAs and possibly to interact with each other. DHX30 binding affinity to CG-motif RNAs was quantified in vitro by means of ALPHA technology, confirming a strong binding of the protein towards the consensus motif. The functional role of the proteins was assessed by performing a stable knockdown of either PCBP2 or DHX30 in HCT116 cells via shRNA. In a PCBP2 downregulated context and to a higher extent after DHX30 silencing, the expression of CG-motif RNAs showed enhanced translation, assessed by RNA-sequencing of the polysome-bound mRNAs and confirmed by the luciferase assay, and via qPCR on a selected mRNAs. Analysis of apoptotic cells by annexin V staining in HCT116 shDHX30 cells exposed to Nutlin-3 for 48 hours confirmed the expectations from previous experiments, establishing a role for DHX30 in the translation control of CG-motif-containing pro-apoptotic mRNAs. MYH9 was tested in SJSA1, being the interactor identified in this cell line from mass spectrometry. Its silencing resulted in the enhanced translation of the luciferase reporter and increased propensity towards cell death. We expanded our model by silencing DHX30 in U2OS cells, which are refractory to apoptosis after Nutlin-3 treatment but share the tissue of origin with SJSA1. Although the silenced clones did not fully recapitulate the phenotype, enhanced expression of CG-motif containing RNAs was observed, resulting in lower vitality of the cells Nutlin-3 exposure. Instead, overexpression of DHX30 in SJSA1 partially rescued the phenotype, lowering the expression of CG-motif containing targets. Overall, we demonstrate how post-transcriptional gene regulation can shape p53-dependent responses in cell lines. We identified a combination of cis-acting (CG-motif) and trans-acting factors (RNA binding proteins) that modulate polysome-loading of pro-apoptotic mRNAs resulting in divergent phenotypic outcomes to Nutlin-3 treatment. We have studied the role of the RNA helicase DHX30 in inhibiting translation of CG-motif containing RNAs, resulting in the survival of cells where this protein is highly expressed. We have optimized an in-vitro assay based on ALPHA technology that can be used to screen for small molecule inhibitors of DHX30-RNA interactions in the perspective of developing a combined treatment with non-genotoxic p53 activation with Nutlin-3 or derivatives to promote cell death over cell cycle arrest in cancer cells.

Beyond transcription: p53-dependent cell death response mediated by DHX30 / Rizzotto, Dario. - (2019 Oct 28), pp. 1-106. [10.15168/11572_243282]

Beyond transcription: p53-dependent cell death response mediated by DHX30

Rizzotto, Dario
2019

Abstract

Given its role as “guardian of the genome”, the protein p53 has been extensively studied in the cancer research field, with the aim of exploiting its potential in cancer therapies. Nutlin-3 can be considered the prototype molecule able to activate WT p53 by inhibiting the interaction with its negative regulator MDM2. Previous evidence coming from literature and experiments performed by other members of the laboratory showed how cell lines undergo different responses when treated with Nutlin-3. Specifically, colon cancer-derived HCT116 cells undergo cell cycle arrest when treated with 10µM of Nutlin-3a for 48 hours, while SJSA1 (osteosarcoma-derived) undergo massive apoptosis. Transcription regulation alone cannot account for the antithetic behaviour of the cell lines since apoptotic factors are transcriptionally modulated in both. Conversely, the analysis of polysome-bound mRNAs revealed a core of differentially regulated genes that share a CG-motif in the 3’UTR whose expression is enhanced in Nutlin-3-treated SJSA1 cells. A luciferase-based assay confirmed that the addition of this motif to a 3’UTR is sufficient to enhance translation of the reporter gene in SJSA1 but not in HCT116 cells treated with Nutlin-3. Analysis of the interactors of the CG-motif by pulldown followed by mass spectrometry revealed a core of interacting RBPs shared between the two cell lines and two specific interactors, DHX30 and MYH9. On the other hand, since the motif was showing a biased composition towards cytosines, we analysed the expression of PCBP family proteins, revealing a differential expression of PCBP2 between HCT116 and SJSA1. The role of PCBP2 and DHX30 was tested in HCT116 cells, being the cell line expressing higher levels of both RBPs. We confirmed the ability of these proteins to interact with CG-motif containing RNAs and possibly to interact with each other. DHX30 binding affinity to CG-motif RNAs was quantified in vitro by means of ALPHA technology, confirming a strong binding of the protein towards the consensus motif. The functional role of the proteins was assessed by performing a stable knockdown of either PCBP2 or DHX30 in HCT116 cells via shRNA. In a PCBP2 downregulated context and to a higher extent after DHX30 silencing, the expression of CG-motif RNAs showed enhanced translation, assessed by RNA-sequencing of the polysome-bound mRNAs and confirmed by the luciferase assay, and via qPCR on a selected mRNAs. Analysis of apoptotic cells by annexin V staining in HCT116 shDHX30 cells exposed to Nutlin-3 for 48 hours confirmed the expectations from previous experiments, establishing a role for DHX30 in the translation control of CG-motif-containing pro-apoptotic mRNAs. MYH9 was tested in SJSA1, being the interactor identified in this cell line from mass spectrometry. Its silencing resulted in the enhanced translation of the luciferase reporter and increased propensity towards cell death. We expanded our model by silencing DHX30 in U2OS cells, which are refractory to apoptosis after Nutlin-3 treatment but share the tissue of origin with SJSA1. Although the silenced clones did not fully recapitulate the phenotype, enhanced expression of CG-motif containing RNAs was observed, resulting in lower vitality of the cells Nutlin-3 exposure. Instead, overexpression of DHX30 in SJSA1 partially rescued the phenotype, lowering the expression of CG-motif containing targets. Overall, we demonstrate how post-transcriptional gene regulation can shape p53-dependent responses in cell lines. We identified a combination of cis-acting (CG-motif) and trans-acting factors (RNA binding proteins) that modulate polysome-loading of pro-apoptotic mRNAs resulting in divergent phenotypic outcomes to Nutlin-3 treatment. We have studied the role of the RNA helicase DHX30 in inhibiting translation of CG-motif containing RNAs, resulting in the survival of cells where this protein is highly expressed. We have optimized an in-vitro assay based on ALPHA technology that can be used to screen for small molecule inhibitors of DHX30-RNA interactions in the perspective of developing a combined treatment with non-genotoxic p53 activation with Nutlin-3 or derivatives to promote cell death over cell cycle arrest in cancer cells.
XXXI
2017-2018
CIBIO (29/10/12-)
Biomolecular Sciences
Inga, Alberto
no
Inglese
File in questo prodotto:
File Dimensione Formato  
Rizzotto_Thesis_2.1_IRIS_Final.pdf

embargo fino al 28/10/2021

Tipologia: Tesi di dottorato (Doctoral Thesis)
Licenza: Tutti i diritti riservati (All rights reserved)
Dimensione 21.85 MB
Formato Adobe PDF
21.85 MB Adobe PDF Visualizza/Apri

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11572/243282
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact