Mark T. Boyd

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.

A Novel Cellular Protein (MTBP) Binds to MDM2 and Induces a G1 Arrest That Is Suppressed by MDM2

The MDM2 protein, through its interaction with p53, plays an important role in the regulation of the G1 checkpoint of the cell cycle. In addition to binding to and inhibiting the transcriptional activation function of the p53 protein, MDM2 binds, inter alia, to RB and the E2F-1·DP-1 complex and in so doing may promote progression of cells into S phase. Mice transgenic for Mdm2 possess cells that have cell cycle regulation defects and develop an altered tumor profile independent of their p53 status. MDM2 also blocks the growth inhibitory effects of transforming growth factor-β1 in a p53-independent manner. We show here that a novel growth regulatory molecule is also the target of MDM2-mediated inhibition. Using a yeast two-hybrid screen, we have identified a gene that encodes a novel cellular protein (MTBP) that binds to MDM2. MTBP can induce G1 arrest, which in turn can be blocked by MDM2. Our results suggest the existence of another growth control pathway that may be regulated, at least in part, by MDM2.

Expression of the chemokines MCP-1/JE and cytokine-induced neutrophil chemoattractant in early acute pancreatitis

Introduction Inflammatory mediators play a critical role in acute pancreatitis. The precise role played by members of the chemokine family remains unclear. Aims To investigate the expression of the CC chemokine monocyte chemotactic protein (MCP)-1/JE and the CXC chemokine cytokine-induced neutrophil chemoattractant (CINC) in early acute pancreatitis. Methodology Pancreatitis was induced in rats, either by intraperitoneal injection of cerulein or by infusion of 5% sodium taurocholate into the pancreatic duct. Expression of MCP-1/JE and CINC in pancreas and plasma was determined by immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), Northern analysis, and quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). Results Following induction of acute pancreatitis, MCP-1/JE and CINC immunoreactivity was seen in acinar cells. Infiltrating neutrophils were strongly immunolabeled with an anti-MCP-1/JE antibody, whereas macrophages reacted strongly with an antibody to CINC. Northern analysis and quantitative real-time RT-PCR demonstrated upregulation of MCP-1/JE and CINC mRNA levels in pancreatic tissue. Plasma MCP-1 levels were significantly increased after 6 hours in the cerulein hyperstimulation model (2,444 ± 93 &mgr;g/mL versus control, 1,853 ± 262 &mgr;g/mL;p < 0.05). Plasma CINC levels were significantly increased after 6 hours in the cerulein hyperstimulation model (1,680 ± 134 &mgr;g/mL versus control, 725 ± 128 &mgr;g/mL;p < 0.005) and after 3 hours in the bile salt infusion model (6,663 ± 1,405 &mgr;g/mL versus control, 2,339 ± 800 &mgr;g/mL;p < 0.05). Conclusion CINC and MCP-1/JE may be early mediators of the inflammatory response in acute pancreatitis.

A Systematic Review of p53 as a Prognostic Factor of Survival in Squamous Cell Carcinoma of the Four Main Anatomical Subsites of the Head and Neck

Objectives: To summarize existing evidence about whether the presence of mutant or upregulated p53 is a prognostic factor for patients presenting with squamous cell carcinoma arising from the larynx, oropharynx, hypopharynx, or oral cavity. Method: Relevant articles were identified using strict criteria for systematic searches. Associations between mutant or upregulated p53 versus wild-type or low/undetectable p53 in relation to overall survival and DFS were summarized by extracting or deriving hazard ratio (HR) estimates. Random-effects meta-analyses were used to account for between-study heterogeneity and to summarize the effect of p53 across studies. Results: The meta-analyses gave a statistically significant pooled HR for overall survival in oral cavity [pooled HR, 1.48; 95% confidence interval, (95% CI), 1.03-2.11], and for disease-free survival in oral cavity (pooled HR, 1.47; 95% CI, 1.12-1.93) and in oropharynx (pooled HR, 0.45; 95% CI, 0.27-0.73). Despite attempts to limit it, between-study heterogeneity was large in the majority of meta-analyses and the prognostic value of p53 was generally inconsistent and inconclusive across studies. Conclusion: The meta-analysis results highlight that current evidence about the prognostic value of p53 in patients with squamous cell carcinaoma of the head and neck is inconclusive. Large heterogeneity exists across studies in study-level and patient-level characteristics, making it difficult to ascertain a clear picture. Future studies are required in which p53 expression is investigated in a more standardized and biologically informative manner. In particular, prospectively planned individual patient data meta-analyses are needed to establish the prognostic importance of p53 for specific subgroups of patients undergoing specific treatments. Cancer Epidemiol Biomarkers Prev; 19(2); 574–87

p53 and MDM2 in Renal Cell Carcinoma: Biomarkers for Disease Progression and Future Therapeutic Targets?

Renal cell carcinoma (RCC) is the most common type of kidney cancer and follows an unpredictable disease course. To improve prognostication, a better understanding of critical genes associated with disease progression is required. The objective of this review was to focus attention on 2 such genes, p53 and murine double minute 2 (MDM2), and to provide a comprehensive summary and critical analysis of the literature regarding these genes in RCC. Information was compiled by searching the PubMed database for articles that were published or e‐published up to April 1, 2009. Search terms included renal cancer, renal cell carcinoma, p53, and MDM2. Full articles and any supplementary data were examined; and, when appropriate, references were checked for additional material. All studies that described assessment of p53 and/or MDM2 in renal cancer were included. The authors concluded that increased p53 expression, but not p53 mutation, is associated with reduced overall survival/more rapid disease progression in RCC. There also was evidence that MDM2 up‐regulation is associated with decreased disease‐specific survival. Two features of RCC stood out as unusual and will require further investigation. First, increased p53 expression is tightly linked with increased MDM2 expression; and, second, patients who have tumors that display increased p53 and MDM2 expression may have the poorest overall survival. Because there was no evidence to support the conclusion that p53 mutation is associated with poorer survival, it seemed clear that increased p53 expression in RCC occurs independent of mutation. Further investigation of the mechanisms leading to increased p53/MDM2 expression in RCC may lead to improved prognostication and to the identification of novel therapeutic interventions. Cancer 2010.

Regulation of p53 and MDM2 activity by MTBP.

ABSTRACT 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 MDM2s 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 γ-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.

The nucleolus directly regulates p53 export and degradation

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

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.

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

The incidence of human papillomavirus (HPV)‐driven disease beyond the oropharynx varies greatly in the reported literature.

MDM2 Regulates Dihydrofolate Reductase Activity through Monoubiquitination

MDM2 is a ubiquitin ligase that is best known for its essential function in the negative regulation of p53. In addition, MDM2 expression is associated with tumor progression in a number of common cancers, and in some cases, this has been shown to be independent of p53 status. MDM2 has been shown to promote the degradation of a number of other proteins involved in the regulation of normal cell growth and proliferation, including MDM4 and RB1. Here, we describe the identification of a novel substrate for the MDM2 ubiquitin ligase: dihydrofolate reductase (DHFR). MDM2 binds directly to DHFR and catalyses its monoubiquitination and not its polyubiquitination. In addition, MDM2 expression reduces DHFR activity in a p53-independent manner, but has no effect upon the steady-state level of expression of DHFR. We show that changes in MDM2 expression alter folate metabolism in cells as evidenced by MDM2-dependent alteration in the sensitivity of cells to the antifolate drug methotrexate. Furthermore, we show that the ability of MDM2 to inhibit DHFR activity depends upon an intact MDM2 RING finger. Our studies provide for the first time a link between MDM2, an oncogene with a critical ubiquitin ligase activity and a vital one-carbon donor pathway involved in epigenetic regulation, and DNA metabolism, which has wide ranging implications for both cell biology and tumor development.