The nucleolus directly regulates p53 export and degradation

Nucleoli directly regulate p53 export and degradation rather than simply sequestering p53 regulatory factors

Misregulation of DNA damage repair pathways in HPV-positive head and neck squamous cell carcinoma contributes to cellular radiosensitivity

Patients with human papillomavirus type 16 (HPV)-associated oropharyngealsquamous cell carcinomas (OPSCC) display increased sensitivity to radiotherapy andimproved survival rates in comparison to HPV-negative forms of the disease. Howeverthe cellular mechanisms responsible for this characteristic difference are unclear.Here, we have investigated the contribution of DNA damage repair pathways to thein vitro radiosensitivity of OPSCC cell lines. We demonstrate that two HPV-positiveOPSCC cells are indeed more radiosensitive than two HPV-negative OPSCC cells, whichcorrelates with reduced efficiency for the repair of ionising radiation (IR)-inducedDNA double strand breaks (DSB). Interestingly, we show that HPV-positive OPSCCcells consequently have upregulated levels of the proteins XRCC1, DNA polymerase β,PNKP and PARP-1 which are involved in base excision repair (BER) and single strandbreak (SSB) repair. This translates to an increased capacity and efficiency for therepair of DNA base damage and SSBs in these cells. In addition, we demonstratethat HPV-positive but interestingly more so HPV-negative OPSCC display increasedradiosensitivity in combination with the PARP inhibitor olaparib. This suggests thatPARP inhibition in combination with radiotherapy may be an effective treatmentfor both forms of OPSCC, particularly for HPV-negative OPSCC which is relativelyradioresistant

CLL Exosomes Modulate the Transcriptome and Behaviour of Recipient Stromal Cells and Are Selectively Enriched in miR-202-3p

Bi-directional communication with the microenvironment is essential for homing and survival of cancer cells with implications for disease biology and behaviour. In chronic lymphocytic leukemia (CLL), the role of the microenvironment on malignant cell behaviour is well described. However, how CLL cells engage and recruit nurturing cells is poorly characterised. Here we demonstrate that CLL cells secrete exosomes that are nanovesicles originating from the fusion of multivesicular bodies with the plasma membrane, to shuttle proteins, lipids, microRNAs (miR) and mRNAs to recipient cells. We characterise and confirm the size (50-100 nm) and identity of the CLL-derived exosomes by Electron microscopy (EM), Atomic force microscopy (AFM), flow cytometry and western blotting using both exosome- and CLL-specific markers. Incubation of CLL-exosomes, derived either from cell culture supernatants or from patient plasma, with human stromal cells shows that they are readily taken up into endosomes, and induce expression of genes such as c-fos and ATM as well as enhance proliferation of recipient HS-5 cells. Furthermore, we show that CLL exosomes encapsulate abundant small RNAs and are enriched in certain miRs and specifically hsa-miR-202-3p. We suggest that such specific packaging of miR-202-3p into exosomes results in enhanced expression of 'suppressor of fused' (Sufu), a Hedgehog (Hh) signalling intermediate, in the parental CLL cells. Thus, our data show that CLL cells secrete exosomes that alter the transcriptome and behaviour of recipient cells. Such communication with microenvironment is likely to have an important role in CLL disease biology

Targeting OGG1 and PARG radiosensitises head and neck cancer cells to high-LET protons through complex DNA damage persistence

Complex DNA damage (CDD), containing two or more DNA lesions within one or two DNA helical turns, is a signature of ionising radiation (IR) and contributes significantly to the therapeutic effect through cell killing. The levels and complexity of CDD increases with linear energy transfer (LET), however, the specific cellular response to this type of DNA damage and the critical proteins essential for repair of CDD is currently unclear. We performed an siRNA screen of ~240 DNA damage response proteins to identify those specifically involved in controlling cell survival in response to high-LET protons at the Bragg peak, compared to low-LET entrance dose protons which differ in the amount of CDD produced. From this, we subsequently validated that depletion of 8-oxoguanine DNA glycosylase (OGG1) and poly(ADP-ribose) glycohydrolase (PARG) in HeLa and head and neck cancer cells leads to significantly increased cellular radiosensitivity specifically following high-LET protons, whilst no effect was observed after low-LET protons and X-rays. We subsequently confirmed that OGG1 and PARG are both required for efficient CDD repair post-irradiation with high-LET protons. Importantly, these results were also recapitulated using specific inhibitors for OGG1 (TH5487) and PARG (PDD00017273). Our results suggest OGG1 and PARG play a fundamental role in the cellular response to CDD and indicate that targeting these enzymes could represent a promising therapeutic strategy for the treatment of head and neck cancers following high-LET radiation

TP53 mutations in head and neck cancer cells determine the Warburg phenotypic switch creating metabolic vulnerabilities and therapeutic opportunities for stratified therapies

Patients with mutated TP53 have been identified as having comparatively poor outcomes compared to those retaining wild-type p53 in many cancers, including squamous cell carcinomas of the head and neck (SCCHN). We have examined the role of p53 in regulation of metabolism in SCCHN cells and find that loss of p53 function determines the Warburg effect in these cells. Moreover, this metabolic adaptation to loss of p53 function creates an Achilles’ heel for tumour cells that can be exploited for potential therapeutic benefit. Specifically, cells lacking normal wild-type p53 function, whether through mutation or RNAi-mediated downregulation, display a lack of metabolic flexibility, becoming more dependent on glycolysis and losing the ability to increase energy production from oxidative phosphorylation. Thus, cells that have compromised p53 function can be sensitised to ionizing radiation by pre-treatment with a glycolytic inhibitor. These results demonstrate the deterministic role of p53 in regulating energy metabolism and provide proof of principle evidence for an opportunity for patient stratification based on p53 status that can be exploited therapeutically using current standard of care treatment with ionising radiation

Non-activated p53 to-localizes with sites of transcription within both the nucleoplasm and the nucleolus

The p53 tumour suppressor functions as a sensor of genotoxic stress and, once activated, induces cell growth arrest or apoptosis. The precise intranuclear localization of latent p53 protein in non-stressed cells is unknown. Such information is essential in order to understand how relatively few molecules of p53 can detect and respond to DNA damage. Here we present the first detailed supramolecular localization of p53 in the nuclei of cells under normal conditions of growth. We show that soluble, non-bound p53 is released by permeabilization leaving structurally bound p53 in both the nucleus and nucleolus. In situ biochemical studies reveal (i) that nuclear-bound p53 is tethered by RNA (directly or indirectly) and (ii) that a sub-population of nuclear-bound p53 co-localizes with sites of RNA synthesis. Transcriptional co-localization appeared to be independent of p53 conformation but dependent upon its quaternary structure. In the nucleolus p53 was observed at sites of rRNA synthesis and also adjacent to such sites. In contrast, nucleolar hdm-2 (shown by others to complex p53 and 5S RNA) was excluded from sites of rRNA synthesis. Our discovery that p53 is physically linked with sites of transcription may explain how relatively few p53 protein molecules can monitor genetic stress and respond preferentially to damage of actively transcribed genes.</p