RASSF1A–LATS1 signalling stabilizes replication forks by restricting CDK2-mediated phosphorylation of BRCA2

Genomic instability is a key hallmark of cancer leading to tumour heterogeneity and therapeutic resistance. ​BRCA2 has a fundamental role in error-free DNA repair but also sustains genome integrity by promoting ​RAD51 nucleofilament formation at stalled replication forks. ​CDK2 phosphorylates ​BRCA2 (pS3291-​BRCA2) to limit stabilizing contacts with polymerized ​RAD51; however, how replication stress modulates ​CDK2 activity and whether loss of pS3291-​BRCA2 regulation results in genomic instability of tumours are not known. Here we demonstrate that the Hippo pathway kinase ​LATS1 interacts with ​CDK2 in response to genotoxic stress to constrain pS3291-​BRCA2 and support ​RAD51 nucleofilaments, thereby maintaining genomic fidelity during replication stalling. We also show that ​LATS1 forms part of an ​ATR-mediated response to replication stress that requires the tumour suppressor ​RASSF1A. Importantly, perturbation of the ​ATR–​RASSF1A–​LATS1 signalling axis leads to genomic defects associated with loss of ​BRCA2 function and contributes to genomic instability and ‘BRCA-ness’ in lung cancers

The role of LATS1 in DNA damage signalling

Genomic DNA is constantly exposed to assaults, which if not dealt with, can lead to genomic instability and carcinogenesis. In response to stress including either Ionising Radiation (IR) or replication stress, ATM and ATR promote the activation of cell cycle checkpoints and initiate repair of DNA damage. Recent studies have revealed that ATM signalling can activate LATS1 via a cascade through RASSF1A and MST2. LATS1 is a tumour suppressor, which forms a barrier to carcinogenesis restricting cell proliferation and promoting apoptosis by stabilising a YAP/p73 transcriptional complex, hence upregulating p73 responsive genes. LATS1 is inactivated through promoter hypermethylation in a number of cancer types including breast cancer and soft tissue sarcoma. This research project seeks to define the mechanism through which LATS1 is involved in IR-induced DNA damage signalling. The data presented in this thesis shows that LATS1 controls CDK2 and regulates phosphorylation of S3291 on BRCA2. Cells lacking LATS1 exhibited enhanced accumulation of damage-induced Rad51 foci leading to cell cycle arrest at the G2/M checkpoint. Furthermore, the data presented here suggests that LATS1 may not be required for homologous recombination. This work supports the hypothesis that LATS1 inhibits CDK2-dependent phosphorylation of BRCA2 at S3291, hence protecting stalled replication forks from nucleolytic degradation.</p

The role of LATS1 in DNA damage signalling

Genomic DNA is constantly exposed to assaults, which if not dealt with, can lead to genomic instability and carcinogenesis. In response to stress including either Ionising Radiation (IR) or replication stress, ATM and ATR promote the activation of cell cycle checkpoints and initiate repair of DNA damage. Recent studies have revealed that ATM signalling can activate LATS 1 via a cascade through RASSF1A and MST2. LATS1 is a tumour suppressor, which forms a barrier to carcinogenesis restricting cell proliferation and promoting apoptosis by stabilising a YAP/p73 transcriptional complex, hence upregulating p73 responsive genes. LATS1 is inactivated through promoter hypermethylation in a number of cancer types including breast cancer and soft tissue sarcoma. This research project seeks to define the mechanism through which LATS1 is involved in IR-induced DNA damage signalling. The data presented in this thesis shows that LATS1 controls CDK2 and regulates phosphorylation of S3291 on BRCA2. Cells lacking LATS1 exhibited enhanced accumulation of damage-induced Rad51 foci leading to cell cycle arrest at the G2/M checkpoint. Furthermore, the data presented here suggests that LATS 1 may not be required for homologous recombination. This work supports the hypothesis that LATS1 inhibits CDK2-dependent phosphorylation of BRCA2 at S3291, hence protecting stalled replication forks from nucleolytic degradationEThOS - Electronic Theses Online ServiceGBUnited Kingdo

The role of LATS1 in DNA damage signalling

Genomic DNA is constantly exposed to assaults, which if not dealt with, can lead to genomic instability and carcinogenesis. In response to stress including either Ionising Radiation (IR) or replication stress, ATM and ATR promote the activation of cell cycle checkpoints and initiate repair of DNA damage. Recent studies have revealed that ATM signalling can activate LATS 1 via a cascade through RASSF1A and MST2. LATS1 is a tumour suppressor, which forms a barrier to carcinogenesis restricting cell proliferation and promoting apoptosis by stabilising a YAP/p73 transcriptional complex, hence upregulating p73 responsive genes. LATS1 is inactivated through promoter hypermethylation in a number of cancer types including breast cancer and soft tissue sarcoma. This research project seeks to define the mechanism through which LATS1 is involved in IR-induced DNA damage signalling. The data presented in this thesis shows that LATS1 controls CDK2 and regulates phosphorylation of S3291 on BRCA2. Cells lacking LATS1 exhibited enhanced accumulation of damage-induced Rad51 foci leading to cell cycle arrest at the G2/M checkpoint. Furthermore, the data presented here suggests that LATS 1 may not be required for homologous recombination. This work supports the hypothesis that LATS1 inhibits CDK2-dependent phosphorylation of BRCA2 at S3291, hence protecting stalled replication forks from nucleolytic degradationEThOS - Electronic Theses Online ServiceGBUnited Kingdo

The role of LATS1 in DNA damage signalling

Genomic DNA is constantly exposed to assaults, which if not dealt with, can lead to genomic instability and carcinogenesis. In response to stress including either Ionising Radiation (IR) or replication stress, ATM and ATR promote the activation of cell cycle checkpoints and initiate repair of DNA damage. Recent studies have revealed that ATM signalling can activate LATS1 via a cascade through RASSF1A and MST2. LATS1 is a tumour suppressor, which forms a barrier to carcinogenesis restricting cell proliferation and promoting apoptosis by stabilising a YAP/p73 transcriptional complex, hence upregulating p73 responsive genes. LATS1 is inactivated through promoter hypermethylation in a number of cancer types including breast cancer and soft tissue sarcoma. This research project seeks to define the mechanism through which LATS1 is involved in IR-induced DNA damage signalling. The data presented in this thesis shows that LATS1 controls CDK2 and regulates phosphorylation of S3291 on BRCA2. Cells lacking LATS1 exhibited enhanced accumulation of damage-induced Rad51 foci leading to cell cycle arrest at the G2/M checkpoint. Furthermore, the data presented here suggests that LATS1 may not be required for homologous recombination. This work supports the hypothesis that LATS1 inhibits CDK2-dependent phosphorylation of BRCA2 at S3291, hence protecting stalled replication forks from nucleolytic degradation.This thesis is not currently available via ORA

Radiation-Induced Hypothyroidism in Patients with Oropharyngeal Cancer Treated with IMRT: Independent and External Validation of Five Normal Tissue Complication Probability Models

We aimed to externally validate five normal tissue complication probability (NTCP) models for radiation-induced hypothyroidism (RIHT) in a prospectively recruited cohort of 108 patients with oropharyngeal cancer (OPC). NTCP scores were calculated using original published formulas. Plasma thyrotropin (TSH) level was additionally assessed in the short-term after RT. After a median of 28 months of follow-up, thirty one (28.7%) patients developed RIHT. Thyroid mean dose and thyroid volume were significant predictors of RIHT: odds ratio equal to 1.11 (95% CI 1.03&ndash;1.19) for mean thyroid dose and 0.87 (95%CI 0.81&ndash;0.93) for thyroid volume in univariate analyses. Two of the evaluated NTCP models, published by R&oslash;njom et al. and by Boomsma et al., had satisfactory performance with accuracies of 0.87 (95%CI 0.79&ndash;0.93) and 0.84 (95%CI: 0.76&ndash;0.91), respectively. Three remaining models, by Cella et al., Bakhshandeh et al. and Vogelius et al., performed significantly worse, overestimating the risk of RIHT in this patient cohort. A short-term TSH level change relative to baseline was not indicative of RIHT development in the follow-up (OR 0.96, 95%CI: 0.65&ndash;1.42, p = 0.825). In conclusion, the models by R&oslash;njom et al. and by Boomsma et al. demonstrated external validity and feasibility for long-term prediction of RIHT in survivors of OPC treated with Intensity-Modulated Radiation Therapy (IMRT)