GC-MS Techniques Investigating Potential Biomarkers of Dying in the Last Weeks with Lung Cancer.

Predicting when a patient with advanced cancer is dying is a challenge and currently no prognostic test is available. We hypothesised that a dying process from cancer is associated with metabolic changes and specifically with changes in volatile organic compounds (VOCs). We analysed urine from patients with lung cancer in the last weeks of life by headspace gas chromatography mass spectrometry. Urine was acidified or alkalinised before analysis. VOC changes in the last weeks of life were identified using univariate, multivariate and linear regression analysis; 12 VOCs increased (11 from the acid dataset, 2 from the alkali dataset) and 25 VOCs decreased (23 from the acid dataset and 3 from the alkali dataset). A Cox Lasso prediction model using 8 VOCs predicted dying with an AUC of 0.77, 0.78 and 0.85 at 30, 20 and 10 days and stratified patients into a low (median 10 days), medium (median 50 days) or high risk of survival. Our data supports the hypothesis there are specific metabolic changes associated with the dying. The VOCs identified are potential biomarkers of dying in lung cancer and could be used as a tool to provide additional prognostic information to inform expert clinician judgement and subsequent decision making.

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.

The unfolded protein response regulator GRP78 is a novel predictive biomarker in colorectal cancer.

Adjuvant fluoropyrimidine-based (5-FU) chemotherapy is a mainstay of treatment for colorectal cancer (CRC), but only provides benefit for a subset of patients. To improve stratification we examined (for the first time in CRC), whether analysis of GRP78 expression provides a predictive biomarker and performed functional studies to examine the role of GRP78 in sensitivity to 5-FU. 396 CRC patient samples were collected in a prospective uniform manner and GRP78 expression was determined by immunohistochemistry on tissue microarrays using a well-validated antibody. Expression was correlated with clinicopathological parameters and survival. The role of GRP78 in 5-FU sensitivity was examined in CRC cells using siRNA, drug inhibition and flow cytometry. GRP78 expression was significantly elevated in cancer tissue (p < 0.0001), and correlated with depth of invasion (p = 0.029) and stage (p = 0.032). Increased overall 5-year survival was associated with high GRP78 expression (p = 0.036). Patients with stage II cancers treated by surgery alone, with high GRP78 also had improved survival (71% v 50%; p = 0.032). Stage III patients with high GRP78 showed significant benefit from adjuvant chemotherapy (52% vs. 28%; p = 0.026), whereas patients with low GRP78 failed to benefit (28% vs. 32%; p = 0.805). Low GRP78 was an independent prognostic indicator of reduced overall 5-year survival (p = 0.004; HR = 1.551; 95%CI 1.155-2.082). In vitro, inhibition of GRP78 reduces apoptosis in response to 5-FU in p53 wild-type cells. GRP78 expression may provide a simple additional risk stratification to inform the adjuvant treatment of CRC and future studies should combine analysis with determination of p53 status.

Whole genome DNA methylation and mutational profiles identify novel changes in proliferative verrucous leukoplakia.

OBJECTIVE: Proliferative verrucous leukoplakia (PVL) is a rare form of oral leukoplakia with a relatively high transformation rate resulting in oral squamous cell carcinoma (OSCC). Molecular analysis of PVL at the genome level is limited and has only identified molecular similarities between PVL and OSCC. However, the clinical profile of PVL suggests that molecular differences may be more important. STUDY DESIGN: Whole exome sequencing of 5 PVL-associated OSCC (PVL-OSCC) and paired blood samples was used to identify somatic mutations common to the tumors. Whole methylome analysis of samples from 4 PVL-associated OSCC and 3 OSCC of non-PVL origin samples was conducted to explore differential methylation. RESULTS: In contrast to conventional OSCC, PVL-associated OSCC showed infrequent TP53 mutation and altered spectra of PIK3CA and NOTCH1 mutations. Unsupervised hierarchical clustering identified 63 probes that discriminated between PVL-associated OSCC and OSCC of non-PVL origin. Differences in methylation were most significant for divalent metal ion transport, particularly calcium movement. CONCLUSIONS: Specific differences in mutation and methylation profiles between PVL-derived OSCC and OSCC of non-PVL origin suggest differences in their transformation pathways. Further studies of early PVL lesions may identify markers of transformation that are also applicable to more common oral premalignant disorders such as oral epithelial dysplasia.

Combined p53 and MDM2 biomarker analysis shows a unique pattern of expression associated with poor prognosis in patients with renal cell carcinoma undergoing radical nephrectomy.

OBJECTIVE: To resolve much debated issues surrounding p53 function, expression and mutation in renal cell carcinoma (RCC), we performed the first study to simultaneously determine p53/MDM2 expression, TP53 mutational status (in p53-positive patients) and outcome in RCC. PATIENTS AND METHODS: In total, 90 specimens obtained from patients with RCC, who were treated by radical nephrectomy, were analyzed by immunohistochemistry for p53 and MDM2 on a tissue microarray, and p53 was functionally and genetically analyzed in p53 positive samples. Outcome analysis was by the Kaplan-Meier method and univariate analysis was used to identify variables for subsequent multivariate analysis of correlations between clinical parameters and biomarker expression. RESULTS: Up-regulation of p53 in RCC is strongly linked with MDM2 up-regulation (P < 0.001). Increased coexpression of p53 and MDM2 identifies those patients with a significantly reduced disease-specific survival by univariate (P= 0.036) and Cox multiple regression analysis (P= 0.027; relative risk, 3.20). Functional (i.e. functional analysis of separated alleles in yeast) and genetic analysis of tumours with increased p53 expression shows that most patients (86%) retain wild-type p53. CONCLUSIONS: Coexpression of p53/MDM2 identifies a subset of patients with poor prognosis, despite all of them having organ-confined disease. Up-regulated p53 is typically wild-type and thus provides a mechanistic explanation for the association between p53 and MDM2 expression: up-regulated wild-type p53 likely promotes the observed MDM2 coexpression. The results obtained in the present study suggest that the p53 pathway is altered in a tissue/disease-specific manner and that therapeutic strategies targeting this pathway should be investigated to determine whether the tumour suppressive function of p53 can be rescued in RCC.

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.

Optimisation of Urine Sample Preparation for Headspace-Solid Phase Microextraction Gas Chromatography-Mass Spectrometry: Altering Sample pH, Sulphuric Acid Concentration and Phase Ratio.

Headspace-solid phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS) can be used to measure volatile organic compounds (VOCs) in human urine. However, there is no widely adopted standardised protocol for the preparation of urine samples for analysis resulting in an inability to compare studies reliably between laboratories. This paper investigated the effect of altering urine sample pH, volume, and vial size for optimising detection of VOCs when using HS-SPME-GC-MS. This is the first, direct comparison of H2SO4, HCl, and NaOH as treatment techniques prior to HS-SPME-GC-MS analysis. Altering urine sample pH indicates that H2SO4 is more effective at optimising detection of VOCs than HCl or NaOH. H2SO4 resulted in a significantly larger mean number of VOCs being identified per sample (on average, 33.5 VOCs to 24.3 in HCl or 12.2 in NaOH treated urine) and more unique VOCs, produced a more diverse range of classes of VOCs, and led to less HS-SPME-GC-MS degradation. We propose that adding 0.2 mL of 2.5 M H2SO4 to 1 mL of urine within a 10 mL headspace vial is the optimal sample preparation prior to HS-SPME-GC-MS analysis. We hope the use of our optimised method for urinary HS-SPME-GC-MS analysis will enhance our understanding of human disease and bolster metabolic biomarker identification.

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.

Regulation of p53 and MDM2 activity by MTBP.

p53 is a critical coordinator of a wide range of stress responses. To facilitate a rapid response to stress, p53 is produced constitutively but is negatively regulated by MDM2. MDM2 can inhibit p53 in multiple independent ways: by binding to its transcription activation domain, inhibiting p53 acetylation, promoting nuclear export, and probably most importantly by promoting proteasomal degradation of p53. The latter is achieved via MDM2's E3 ubiquitin ligase activity harbored within the MDM2 RING finger domain. We have discovered that MTBP promotes MDM2-mediated ubiquitination and degradation of p53 and also MDM2 stabilization in an MDM2 RING finger-dependent manner. Moreover, using small interfering RNA to down-regulate endogenous MTBP in unstressed cells, we have found that MTBP significantly contributes to MDM2-mediated regulation of p53 levels and activity. However, following exposure of cells to UV, but not gamma-irradiation, MTBP is destabilized as part of the coordinated cellular response. Our findings suggest that MTBP differentially regulates the E3 ubiquitin ligase activity of MDM2 towards two of its most critical targets (itself and p53) and in doing so significantly contributes to MDM2-dependent p53 homeostasis in unstressed cells.

Metabolic signature of squamous cell carcinoma of the head and neck: Consequences of TP53 mutation and therapeutic perspectives.

There is a pressing need to identify ways of sensitising squamous cell carcinomas of the head and neck (SCCHN) to the effects of current treatments, both from oncological and functional perspectives. Alteration to cellular metabolism is now widely considered a hallmark of the cancer phenotype; presents a potentially attractive therapeutic target in this regard; and as such has received renewed research interest in recent years. However, whilst metabolic disruption may occur to some degree in all tumours, there is undoubtedly heterogeneity and detailed study of individual tumour types is paramount if effective therapeutic strategies targeting metabolism are to be developed and effectively deployed. In this review we outline current understanding of altered tumour metabolism and how these adaptations promote tumorigenesis generally. We relate this specifically to SCCHN by focusing on several recent key studies specific to SCCHN, and by discussing the role TP53 mutation may play in this metabolic switch, given the fundamental role of this oncogenic event in SCCHN tumorigenesis. Finally, we also offer insight into the potential therapeutic implications this may have in the clinical setting and make recommendations for future study.

p53 regulation and function in renal cell carcinoma.

Loss of p53 function is a critical event in tumor evolution. This occurs through a range of molecular events, typically a missense p53 mutation followed by loss of heterozygosity. In many cancers, there is compelling evidence that cells that can compromise p53 function have a selective advantage. The situation in renal cell carcinoma is unclear. It has recently been suggested that p53 function is unusually compromised in renal carcinoma cells by a novel dominant, MDM2/p14ARF-independent mechanism. This is hard to reconcile with other recent studies that have identified p53 as an important prognostic indicator. Indeed, one of these latter studies found that the best predictor of poor outcome was the presence of high levels of both p53 (usually indicative of p53 mutation) and MDM2. Thus, it is important that we gain a clearer understanding of the regulation of p53 and the role of MDM2 in renal cell cancer. To address this, we have investigated the transcriptional activity of p53 in a panel of renal cell carcinoma cell lines and the contribution of MDM2 and p14ARF to p53 regulation. We have found that p53 is functional in p53 wild-type renal cell carcinoma cells and that this activity is significantly regulated by MDM2 and to a much lesser extent by p14ARF. Moreover, following induction of DNA damage with UV, the p53 response in these cells is intact. Thus, future studies of renal cell carcinoma that focus on p53 and MDM2 and their role in determining disease outcome will be required to create a better understanding of this notoriously difficult to manage disease.

Stimulation of human DNA polymerase epsilon by MDM2.

The human DNA polymerase epsilon catalytic subunit consists of a 140-kDa N-terminal domain that contains the catalytic activity and a 120-kDa C-terminal domain that binds to the other subunits and to exogenous peptides, including PCNA and MDM2. We report here that recombinant human MDM2 purified from insect cells or Escherichia coli stimulated the activity of DNA polymerase epsilon up to 10- and 40-fold, respectively, but not those of DNA polymerase beta or Klenow fragment of E.coli DNA polymerase I. Kinetic studies indicated that MDM2 increased the maximum velocity of the reaction, but did not change substrate affinities. The stimulation depended upon the interaction of the N-terminal 166 amino acid residues of MDM2 with the C-terminal domain of the full-length catalytic subunit, since the deletion of 166 amino acids from N-terminal of MDM2 or the removal of the C-terminal domain of DNA polymerase epsilon by trypsin digestion or competition for binding to it by the addition of excess C-terminal fragment eliminated the stimulation. Since DNA polymerase epsilon appears to be involved in DNA replication, recombination and repair synthesis, we suggest that MDM2 binding to DNA polymerase epsilon might be part of a reconfiguration process that allows DNA polymerase epsilon to associate with repair/recombination proteins in response to DNA damage.

Relative expression of vascular endothelial growth factor isoforms in squamous cell carcinoma of the head and neck.

BACKGROUND: Alternative splicing of the vascular endothelial growth factor (VEGF) gene results in a family of antiangiogenic isoforms (VEGFxxx b), not yet investigated in squamous cell carcinoma of the head and neck (SCCHN). We examined, therefore, the prognostic value of the relative expression of VEGF isoforms in SCCHN. METHODS: A tissue microarray comprising 187 SCCHNs was studied by immunohistochemistry with total VEGF (panVEGF) and VEGFxxx b-specific antibodies, and scored by 2 assessors for intensity and proportion. Scores were combined and expression ratios calculated. RESULTS: No meaningful significant differences were observed between panVEGF, VEGFxxx b, or expression ratio, and presence of lymphatic metastasis, or overall survival. This held true when tumor subsites were analyzed independently and when human papillomavirus (HPV) was accounted for in the oropharyngeal subgroup. CONCLUSION: Differential VEGF isoform expression is not a reliable prognostic biomarker for either the clinically node negative/pathologically node-positive neck or overall survival in pharyngeal and laryngeal SCCHNs.

Comprehensive analysis of matrix metalloproteinase and tissue inhibitor expression in pancreatic cancer: increased expression of matrix metalloproteinase-7 predicts poor survival.

PURPOSE: To enable the design of improved inhibitors of matrix metalloproteinases (MMPs) for the treatment of pancreatic cancer, the expression profiles of a range of MMPs and tissue inhibitors of MMPs (TIMPs) were determined. EXPERIMENTAL DESIGN: Nine MMPs (MMPs 1-3, 7-9, 11, 12, and 14) and three TIMPs (TIMPs 1-3) were examined in up to 75 pancreatic ductal adenocarcinomas and 10 normal pancreata by immunohistochemistry. Eighteen additional pancreatic ductal adenocarcinomas and an additional eight normal pancreata were also analyzed by real-time reverse transcription-PCR and additionally for MMP-15. RESULTS: There was increased expression by immunohistochemistry for MMPs 7, 8, 9, and 11 and TIMP-3 in pancreatic cancer compared with normal pancreas (P < 0.0001, 0.04, 0.0009, 0.005, and 0.0001, respectively). Real-time reverse transcription-PCR showed a significant increase in mRNA levels for MMP-11 in tumor tissue compared with normal pancreatic tissue (P = 0.0005) and also significantly reduced levels of MMP-15 (P = 0.0026). Univariate analysis revealed that survival was reduced by lymph node involvement (P = 0.0007) and increased expression of MMP-7 (P = 0.005) and (for the first time) MMP-11 (P = 0.02) but not reduced by tumor grade, tumor diameter, positive resection margins, adjuvant treatment, or expression of the remaining MMPs and TIMPs. On multivariate analysis, only MMP-7 predicted shortened survival (P < 0.05); however, increased MMP-11 expression was strongly associated with lymph node involvement (P = 0.0073). CONCLUSIONS: We propose that the principle specificity for effective inhibitors of MMPs in pancreatic cancer should be for MMP-7 with secondary specificity against MMP-11. Moreover, these studies indicate that MMP-7 expression is a powerful independent prognostic indicator and potentially of considerable clinical value.

Janus-faces of NME-oncoprotein interactions.

Since the identification of Nm23 (NME1, NME/NM23 nucleoside diphosphate kinase 1) as the first non-metastatic protein, a great deal of research on members of the NME family of proteins has focused on roles in processes implicated in carcinogenesis and particularly their regulation of cellular motility and the process of metastatic spread. To date, there are ten identified members of this family of genes, and these can be dichotomized into groups both taxonomically and by the presence or absence of their nucleoside diphosphate kinase activity with NMEs 1-4 encoding nucleoside diphosphate kinases (NDPKs) and NMEs 5-9 plus RP2 displaying little if any NDPK activity. NMEs are relatively small proteins that can form hetero-oligomers (typically hexamers), and given the apparent genetic redundancy of some NMEs and the number of different isoforms, it is perhaps not surprising that there remains a great deal of uncertainty regarding their function and even more regarding cellular mechanisms of action. Since residues that contribute to NDPK activity span much of the protein, it seems likely that the consequences of NME expression must be mediated through their NDPK activity, through interactions with other structures in cells including protein-protein interactions or through combinations of these. Our goal in this review is to focus on some of the protein-protein interactions that have been identified and to highlight some of the challenges that face this area of research.

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.

Squamous cell carcinoma of the head and neck outside the oropharynx is rarely human papillomavirus related.

OBJECTIVES/HYPOTHESIS: The incidence of human papillomavirus (HPV)-driven disease beyond the oropharynx varies greatly in the reported literature. STUDY DESIGN: Case series. METHODS: Two hundred twenty-one samples were strictly classified to the subsites of oral cavity, larynx, or hypopharynx at the time of primary surgery. Formalin-fixed paraffin-embedded samples were subjected to a validated, tiered, diagnostic algorithm of p16 immunohistochemistry, high-risk HPV in situ hybridization, and quantitative polymerase chain reaction for HPV E6 DNA. An additional 60 oropharyngeal cases acted as an internal biological control. RESULTS: An incidence of 4% of HPV-driven cases was observed across the subsites outside the oropharynx compared to 70% of tumors confined within it. CONCLUSIONS: This is the first reporting of a broad range of nonoropharyngeal HPV rates using this validated diagnostic algorithm. It remains unclear whether patients with HPV-driven disease originating outside the oropharynx enjoy the same survival advantage apparent in those patients with oropharyngeal squamous cell carcinomas. LEVEL OF EVIDENCE: 4 Laryngoscope, 124:2739-2744, 2014.

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.