Nucleolar control of p53: a cellular Achilles' heel and a target for cancer therapy.

Nucleoli perform a crucial cell function, ribosome biogenesis, and of critical relevance to the subject of this review, they are also extremely sensitive to cellular stresses, which can cause loss of function and/or associated structural disruption. In recent years, we have learned that cells take advantage of this stress sensitivity of nucleoli, using them as stress sensors. One major protein regulated by this role of nucleoli is the tumor suppressor p53, which is activated in response to diverse cellular injuries in order to exert its onco-protective effects. Here we discuss a model of nucleolar regulation of p53, which proposes that key steps in the promotion of p53 degradation by the ubiquitin ligase MDM2 occur in nucleoli, thus providing an explanation for the observed link between nucleolar disruption and p53 stability. We review current evidence for this compartmentalization in p53 homeostasis and highlight current limitations of the model. Interestingly, a number of current chemotherapeutic agents capable of inducing a p53 response are likely to do so by targeting nucleolar functions and these compounds may serve to inform further improved therapeutic targeting of nucleoli.

Myoferlin: A Potential Marker of Response to Radiation Therapy and Survival in Locally Advanced Rectal Cancer.

PURPOSE: Patients with locally advanced rectal cancer often require neoadjuvant chemoradiation therapy to downstage the disease, but the response is variable with no predictive biomarkers. We have previously revealed through proteomic profiling that myoferlin is associated with response to radiation therapy. The aims of this study were to further validate this finding and explore the potential for myoferlin to act as a prognostic and/or therapeutic target. METHODS AND MATERIALS: Immunohistochemical analysis of a tissue microarray (TMA) for 111 patients was used to validate the initial proteomic findings. Manipulation of myoferlin was achieved using small interfering RNA, a small molecular inhibitor (wj460), and a CRISPR-Cas9 knockout cell line. Radiosensitization after treatment was assessed using 2-dimensional clonogenic assays, 3-dimensional spheroid models, and patient-derived organoids. Underlying mechanisms were investigated using electrophoresis, immunofluorescence, and immunoblotting. RESULTS: Analysis of both the diagnostic biopsy and tumor resection samples confirmed that low myoferlin expression correlated with a good response to neoadjuvant long-course chemoradiation therapy. High myoferlin expression was associated with spread to local lymph nodes and worse 5-year survival (P = .01; hazard ratio, 3.5; 95% CI, 1.27-10.04). This was externally validated using the Stratification in Colorectal Cancer database. Quantification of myoferlin using immunoblotting in immortalized colorectal cancer cell lines and organoids demonstrated that high myoferlin expression was associated with increased radioresistance. Biological and pharmacologic manipulation of myoferlin resulted in significantly increased radiosensitivity across all cell lines in 2-dimensional and 3-dimensional models. After irradiation, myoferlin knockdown cells had a significantly impaired ability to repair DNA double-strand breaks. This appeared to be mediated via nonhomologous end-joining. CONCLUSIONS: We have confirmed that high expression of myoferlin in rectal cancer is associated with poor response to neoadjuvant therapy and worse long-term survival. Furthermore, the manipulation of myoferlin led to increased radiosensitivity in vitro. This suggests that myoferlin could be targeted to enhance the sensitivity of patients with rectal cancer to radiation therapy, and further work is required.

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.

MDM2 interacts with NME2 (non-metastatic cells 2, protein) and suppresses the ability of NME2 to negatively regulate cell motility.

MDM2 expression, combined with increased p53 expression, is associated with reduced survival in several cancers, but is particularly of interest in renal cell carcinoma (RCC) where evidence suggests the presence of tissue-specific p53/MDM2 pathway defects. We set out to identify MDM2-interacting proteins in renal cells that could act as mediators/targets of MDM2 oncogenic effects in renal cancers. We identified the non-metastatic cells 2, protein; NME2 (NDPK-B, NM23-B/-H2), a nucleoside diphosphate kinase, as an MDM2-interacting protein using both a proteomic-based strategy [affinity chromatography and tandem mass spectrometry [MS/MS] from HEK293 cells] and a yeast two-hybrid screen of a renal carcinoma cell-derived complementary DNA library. The MDM2-NME2 interaction is highly specific, as NME1 (87.5% amino acid identity) does not interact with MDM2 in yeast. Specific NME proteins display well-documented cell motility and metastasis-suppressing activity. We show that NME2 contributes to motility suppression under conditions where MDM2 is expressed at normal physiological/low levels. However, up-regulation of MDM2 in RCC cells abolishes the ability of NME2 to suppress motility. Significantly, when MDM2 expression is down-regulated in these cells using small interfering RNA, the motility-suppressing activity of NME2 is rescued, confirming that MDM2 expression causes the loss of NME2 cell motility regulatory function. Thus MDM2 up-regulation in renal cancer cells can act in a dominant manner to abrogate the function of a potent suppressor of motility and metastasis. Our studies identify a novel protein-protein interaction between MDM2 and NME2, which suggests a mechanism that could explain the link between MDM2 expression and poor patient survival in RCC.

Loss of MTBP expression is associated with reduced survival in a biomarker-defined subset of patients with squamous cell carcinoma of the head and neck.

BACKGROUND: Recent genetic studies have implicated p53 mutation as a significant risk factor for therapeutic failure in squamous cell carcinoma of the head and neck (SCCHN). However, in a recent meta-analysis in the literature of p53 from major anatomical subsites (larynx, oral cavity, oropharynx/hypopharynx), associations between patient survival and p53 status were ambiguous. METHODS: The authors examined a cohort of SCCHNs using a previously developed biomarker combination that likely predicts p53 status based on p53/MDM2 expression levels determined by immunohistochemistry (IHC). In addition, the authors generated and validated an antibody to MTBP (an MDM2 binding protein that alters p53/MDM2 homeostasis and may contribute to metastatic suppression) and have incorporated data for MTBP expression into the current analyses. RESULTS: Analysis of expression data for p53 and MDM2 in 198 SCCHN patient samples revealed that the biomarker combination p53 + ve/MDM2-low (likely indicative of p53 mutation) was significantly associated with reduced overall survival (log-rank P = .035) and was an independent prognostic factor (P = .013; HR, 1.705; 95% CI, 1.12-2.60); thus, these data were compatible with earlier genetic analyses. By using IHC for p53 and MDM2 to dichotomize patients, the authors found that loss of MTBP expression was significantly associated with reduced survival (log-rank P = .004) and was an independent prognostic factor (P = .004; HR, 2.78; 95% CI, 1.39-5.54) in p53 + ve/MDM2-low patients. CONCLUSIONS: These results represent the first examination of MTBP expression in human tissues and provide evidence for a p53 status-dependent role for MTBP in suppressing disease progression in SCCHN patients as well as confirming a role for p53 pathway function in delaying disease progression.

Metabolic Plasticity and Combinatorial Radiosensitisation Strategies in Human Papillomavirus-Positive Squamous Cell Carcinoma of the Head and Neck Cell Lines.

BACKGROUND: A major objective in the management of human papillomavirus (HPV)-positive squamous cell carcinoma of the head and neck (SCCHN) is to reduce long-term functional ramifications while maintaining oncological outcomes. This study examined the metabolic profile of HPV-positive SCCHN and the potential role of anti-metabolic therapeutics to achieve radiosensitisation as a potential means to de-escalate radiation therapy. METHODS: Three established HPV-positive SCCHN cell lines were studied (UM-SCC-104, UPCI:SCC154, and VU-SCC-147), together with a typical TP53 mutant HPV-negative SCCHN cell line (UM-SCC-81B) for comparison. Metabolic profiling was performed using extracellular flux analysis during specifically designed mitochondrial and glycolytic stress tests. Sensitivity to ionising radiation (IR) was evaluated using clonogenic assays following no treatment, or treatment with: 25 mM 2-deoxy-D-glucose (glycolytic inhibitor) alone; 20 mM metformin (electron transport chain inhibitor) alone; or 25 mM 2-deoxy-D-glucose and 20 mM metformin combined. Expression levels of p53 and reporters of p53 function (MDM2, p53, Phospho-p53 [Ser15], TIGAR and p21 [CDKN1A]) were examined by western blotting. RESULTS: HPV-positive SCCHN cell lines exhibited a diverse metabolic phenotype, displaying robust mitochondrial and glycolytic reserve capacities. This metabolic profile, in turn, correlated with IR response following administration of anti-metabolic agents, in that both 2-deoxy-D-glucose and metformin were required to significantly potentiate the effects of IR in these cell lines. CONCLUSIONS: In contrast to our recently published data on HPV-negative SCCHN cells, which display relative glycolytic dependence, HPV-positive SCCHN cells can only be sensitised to IR using a complex anti-metabolic approach targeting both mitochondrial respiration and glycolysis, reflecting their metabolically diverse phenotype. Notionally, this may provide an attractive platform for treatment de-intensification in the clinical setting by facilitating IR dose reduction to minimise the impact of treatment on long-term function.

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.

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 oropharyngeal squamous cell carcinomas (OPSCC) display increased sensitivity to radiotherapy and improved survival rates in comparison to HPV-negative forms of the disease. However the cellular mechanisms responsible for this characteristic difference are unclear. Here, we have investigated the contribution of DNA damage repair pathways to the in vitro radiosensitivity of OPSCC cell lines. We demonstrate that two HPV-positive OPSCC cells are indeed more radiosensitive than two HPV-negative OPSCC cells, which correlates with reduced efficiency for the repair of ionising radiation (IR)-induced DNA double strand breaks (DSB). Interestingly, we show that HPV-positive OPSCC cells consequently have upregulated levels of the proteins XRCC1, DNA polymerase ß, PNKP and PARP-1 which are involved in base excision repair (BER) and single strand break (SSB) repair. This translates to an increased capacity and efficiency for the repair of DNA base damage and SSBs in these cells. In addition, we demonstrate that HPV-positive but interestingly more so HPV-negative OPSCC display increased radiosensitivity in combination with the PARP inhibitor olaparib. This suggests that PARP inhibition in combination with radiotherapy may be an effective treatment for both forms of OPSCC, particularly for HPV-negative OPSCC which is relatively radioresistant.

The interaction of p53 with the nuclear matrix is mediated by F-actin and modulated by DNA damage.

The tumour suppressor protein p53 is localized in the cell nucleus where it serves to initiate cellular responses to a variety of stresses, particularly DNA damage and has the capacity to transactivate stress response genes. An emerging body of evidence indicates that its action is also exerted through direct protein-protein interactions. An approach to understanding p53 function has been to analyse its positioning in relation to nuclear structures and we have shown that p53 can associate with the nuclear matrix. A potential nuclear matrix component for this association is actin. Here we show that p53 interacts with nuclear F-actin and we map the domains involved in this interaction. Using fluorescence resonance energy transfer, we demonstrate that the partition of p53 between F-actin bound and unbound forms is not constant, but is modulated by the presence of DNA damage, which increases binding. Our results indicate that the dynamic interaction of p53 with the nuclear matrix has to be considered for a full understanding of the mechanisms of the p53-mediated cellular response to DNA damage.

UV-C-induced DNA damage leads to p53-dependent nuclear trafficking of PML.

The promyelocytic leukemia protein (PML) is a nuclear phosphoprotein that localizes to distinct domains in the nucleus, described as PML nuclear bodies (PML-NBs). Recent findings indicate that PML regulates the p53 response to oncogenic signals. Here, we define a p53-dependent role for PML in response to DNA damage. We exposed cells to ultraviolet light (UV-C) and imaged the nuclear distribution of PML, p53, and the BLM helicase by confocal microscopy. After DNA damage, PML partially relocated out of the PML-NBs, and colocalized with BLM and p53 at sites of DNA repair. In addition, using the isogenic HCT116 cell lines (p53+/+ and -/-), we show that the redistribution of PML was dependent on functional p53. Western analysis revealed that the level of PML protein remained unaltered after UV-C treatment. These results are consistent with the hypothesis that PML, in conjunction with p53 and BLM, contributes to the cellular response to UV-C-induced DNA damage and its repair.

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.

Gel-based application of siRNA to human epithelial cancer cells induces RNAi-dependent apoptosis.

Gene silencing by RNA interference (RNAi) operates at the level of mRNA that is targeted for destruction with exquisite sequence specificity. In principle, any disease-related mRNA sequence is a putative target for RNAi-based therapeutics. To develop this therapeutic potential, it is necessary to develop ways of inducing RNAi by clinically acceptable delivery procedures. Here, we ask if inducers of RNAi can be delivered to human cells via a gel-based medium. RNAi was induced using synthetic small interfering RNAs (siRNAs), which bypass the need for expression vectors and carry the added bonus of high potency and immediate efficacy. Established cultures of human cells of normal and tumor origin were overlaid with an agarose/liposome/siRNA gel formulation without adverse effects on cell viability or proliferation. Epithelial cancer cells (but not normal human fibroblasts) proved vulnerable to specific siRNAs delivered via the agarose/liposome/siRNA formulation. Moreover, proapoptotic siRNAs induced apoptosis of cervical carcinoma cells (treated with human papillomavirus [HPV] E7 siRNA) and of colorectal carcinoma cells (treated with Bcl-2 siRNA). Thus, we demonstrate successful topical gel-based delivery of inducers of RNAi to human epithelial cancer cells. Topical induction of RNAi opens an important new therapeutic approach for treatment of human diseases, including cervical cancer and other accessible disorders.

Senescence induction in renal carcinoma cells by Nutlin-3: a potential therapeutic strategy based on MDM2 antagonism.

Although the role of p53 as a tumour suppressor in renal cell carcinoma (RCC) is unclear, our recent analysis suggests that increased wild-type p53 protein expression is associated with poor outcome. A growing body of evidence also suggests that p53 expression and increased co-expression of MDM2 are linked with poor prognosis in RCC. We have therefore examined whether an MDM2 antagonist; Nutlin-3, might rescue/increase p53 expression and induce growth inhibition or apoptosis in RCC cells that retain wild-type p53. We show that inhibition of p53 suppression by MDM2 in RCC cells promotes growth arrest and p53-dependent senescence - phenotypes known to mediate p53 tumour suppression in vivo. We propose that future investigations of therapeutic strategies for RCC should incorporate MDM2 antagonism as part of strategies aimed at rescuing/augmenting p53 tumour suppressor function.

The nucleolus directly regulates p53 export and degradation.

The correlation between stress-induced nucleolar disruption and abrogation of p53 degradation is evident after a wide variety of cellular stresses. This link may be caused by steps in p53 regulation occurring in nucleoli, as suggested by some biochemical evidence. Alternatively, nucleolar disruption also causes redistribution of nucleolar proteins, potentially altering their interactions with p53 and/or MDM2. This raises the fundamental question of whether the nucleolus controls p53 directly, i.e., as a site where p53 regulatory processes occur, or indirectly, i.e., by determining the cellular localization of p53/MDM2-interacting factors. In this work, transport experiments based on heterokaryons, photobleaching, and micronucleation demonstrate that p53 regulatory events are directly regulated by nucleoli and are dependent on intact nucleolar structure and function. Subcellular fractionation and nucleolar isolation revealed a distribution of ubiquitylated p53 that supports these findings. In addition, our results indicate that p53 is exported by two pathways: one stress sensitive and one stress insensitive, the latter being regulated by activities present in the nucleolus.

Tissue-specific therapeutic targeting of p53 in cancer: one size does not fit all.

p53, the "guardian of the genome" and the most mutated gene in cancer presents a considerable therapeutic opportunity as well as a challenge. In the past decade, several therapeutic strategies have been developed that aim to take advantage of a wealth of knowledge about p53, including insights into the biology and patho-biology of p53. Nevertheless, considerable challenges remain, not least as a result of tissue- and cancer-specific differences in p53 regulation and/or function. p53 does not act in the same manner in all tissues or in the cancers arising from them. Nor is p53 regulated in the same way in the wide variety of tissues from which cancers develop. Therefore, potential strategies for therapeutic targeting need to be tailored to each tumour/tissue type. This review summarises some of these tissue- and cancer-specific issues to suggest how different strategies are required for cancers arising from different tissues and to illustrate the complexities of therapeutic targeting of p53.

MDM2 promotes cell motility and invasiveness through a RING-finger independent mechanism.

Recent studies connect MDM2 with increased cell motility, invasion and/or metastasis proposing an MDM2-mediated ubiquitylation-dependent mechanism. Interestingly, in renal cell carcinoma (RCC) p53/MDM2 co-expression is associated with reduced survival which is independently linked with metastasis. We therefore investigated whether expression of p53 and/or MDM2 promotes aggressive cell phenotypes. Our data demonstrate that MDM2 promotes increased motility and invasiveness in RCC cells (N.B. similar results are obtained in non-RCC cells). This study shows for the first time both that endogenous MDM2 significantly contributes to cell motility and that this does not depend upon the MDM2 RING-finger, i.e. is independent of ubiquitylation (and NEDDylation). Our data suggest that protein-protein interactions provide a likely mechanistic basis for MDM2-promoted motility which may constitute future therapeutic targets.

Disruption of the nucleolus mediates stabilization of p53 in response to DNA damage and other stresses.

p53 protects against cancer through its capacity to induce cell cycle arrest or apoptosis under a large variety of cellular stresses. It is not known how such diversity of signals can be integrated by a single molecule. However, the literature reveals that a common denominator in all p53-inducing stresses is nucleolar disruption. We thus postulated that the impairment of nucleolar function might stabilize p53 by preventing its degradation. Using micropore irradiation, we demonstrate that large amounts of nuclear DNA damage fail to stabilize p53 unless the nucleolus is also disrupted. Forcing nucleolar disruption by anti-upstream binding factor (UBF) microinjection (in the absence of DNA damage) also causes p53 stabilization. We propose that the nucleolus is a stress sensor responsible for maintenance of low levels of p53, which are automatically elevated as soon as nucleolar function is impaired in response to stress. Our model integrates all known p53-inducing agents and also explains cell cycle-related variations in p53 levels which correlate with established phases of nucleolar assembly/disassembly through the cell cycle.

p53 is a chromatin accessibility factor for nucleotide excision repair of DNA damage.

One of the longest standing problems in DNA repair is how cells relax chromatin in order to make DNA lesions accessible for global nucleotide excision repair (NER). Since chromatin has to be relaxed for efficient lesion detection, the key question is whether chromatin relaxation precedes lesion detection or vice versa. Chromatin accessibility factors have been proposed but not yet identified. Here we show that p53 acts as a chromatin accessibility factor, mediating UV-induced global chromatin relaxation. Using localized subnuclear UV irradiation, we demonstrate that chromatin relaxation is extended over the whole nucleus and that this process requires p53. We show that the sequence for initiation of global NER is as follows: transcription-associated lesion detection; p53-mediated global chromatin relaxation; and global lesion detection. The tumour suppressor p53 is crucial for genomic stability, a role partially explained by its pro-apoptotic capacity. We demonstrate here that p53 is also a fundamental component of DNA repair, playing a direct role in rectifying DNA damage.