MDM2 and MDMX function as key regulators of p53 by binding to its N terminus, inhibiting its transcriptional activity, and promoting degradation. MDM2 and MDMX overexpression or hyperactivation directly contributes to the loss of p53 function during the development of nearly 50% of human cancers. Recent studies showed that disrupting p53-MDM2 and p53-MDMX interactions can lead to robust activation of p53 but also revealed a need to develop novel dual specific or MDMXspecific inhibitors. Using phage display we identified a 12-residue peptide (pDI) with inhibitory activity against MDM2 and MDMX. The co-crystal structures of the pDI and a single mutant derivative (pDI6W) liganded with the N-terminal domains of human MDMX and MDM2 served as the basis for the design of 11 distinct pDI-derivative peptides that were tested for inhibitory potential. The best derivative (termed pDIQ) contained four amino acid substitutions and exhibited a 5-fold increase in potency over the parent peptide against both MDM2 (IC 50 ‫؍‬ 8 nM) and MDMX (IC 50 ‫؍‬ 110 nM). Further structural studies revealed key molecular features enabling the high affinity binding of the pDIQ to these proteins. These include large conformational changes of the pDIQ to reach into a hydrophobic site unique to MDMX. The findings suggest new strategies toward the rational design of small molecule inhibitors efficiently targeting MDMX.The p53 tumor suppressor is a potent inducer of cell cycle arrest, apoptosis, cellular senescence, and innate immunity. It is activated in response to oncogenic transformation, extrinsic stress, and viral infection to protect higher organisms from cancer (1-3). p53 also facilitates maternal reproduction through induction of the growth factor leukemia inhibitory factor (LIF) that promotes embryo implantation (4). p53 activity is kept at minimal levels in unstressed cells by interactions with MDM2 and MDMX. MDM2 is an ubiquitin E3 ligase for p53 and an important regulator of p53 stability by forming a negative feedback loop (5, 6). The MDM2 homolog MDMX also binds to p53 and inhibits p53-dependent transcription (7). Loss of MDM2 or MDMX leads to embryonic lethality (8 -10). Therefore, the expression of MDM2 and the expression of MDMX are both necessary for regulation of p53 during normal development.Genetic or functional inactivation of p53 is an obligatory step during cancer development. In human tumors that retain wild type p53, amplification of MDM2 or MDMX serves as an alternative mechanism of p53 inactivation in a subset of tumors (11,12). Furthermore, MDM2 activity is controlled by the tumor suppressor ARF (alternative reading frame) encoded by the INK4a locus (2). Deletion/epigenetic silencing of ARF occur in most tumors expressing wild type p53, resulting in hyperactive MDM2 and lack of p53 response to oncogenic stress in malignant tumors (13,14). ARF has also been shown to promote MDMX degradation by MDM2 (15). Loss of ARF expression may result in MDMX stabilization that further inactivates p53. Therefore, MDM2 and MDMX are d...

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BackgroundIntraductal papillary mucinous neoplasms (IPMNs) are pancreatic ductal adenocarcinoma (PDAC) precursors. Differentiating between high-risk IPMNs that warrant surgical resection and low-risk IPMNs that can be monitored is a significant clinical problem, and we sought to discover a panel of mi(cro)RNAs that accurately classify IPMN risk status.Methodology/Principal FindingsIn a discovery phase, genome-wide miRNA expression profiling was performed on 28 surgically-resected, pathologically-confirmed IPMNs (19 high-risk, 9 low-risk) using Taqman MicroRNA Arrays. A validation phase was performed in 21 independent IPMNs (13 high-risk, 8 low-risk). We also explored associations between miRNA expression level and various clinical and pathological factors and examined genes and pathways regulated by the identified miRNAs by integrating data from bioinformatic analyses and microarray analysis of miRNA gene targets. Six miRNAs (miR-100, miR-99b, miR-99a, miR-342-3p, miR-126, miR-130a) were down-regulated in high-risk versus low-risk IPMNs and distinguished between groups (P<10−3, area underneath the curve (AUC) = 87%). The same trend was observed in the validation phase (AUC = 74%). Low miR-99b expression was associated with main pancreatic duct involvement (P = 0.021), and serum albumin levels were positively correlated with miR-99a (r = 0.52, P = 0.004) and miR-100 expression (r = 0.49, P = 0.008). Literature, validated miRNA:target gene interactions, and pathway enrichment analysis supported the candidate miRNAs as tumor suppressors and regulators of PDAC development. Microarray analysis revealed that oncogenic targets of miR-130a (ATG2B, MEOX2), miR-342-3p (DNMT1), and miR-126 (IRS-1) were up-regulated in high- versus low-risk IPMNs (P<0.10).ConclusionsThis pilot study highlights miRNAs that may aid in preoperative risk stratification of IPMNs and provides novel insights into miRNA-mediated progression to pancreatic malignancy. The miRNAs identified here and in other recent investigations warrant evaluation in biofluids in a well-powered prospective cohort of individuals newly-diagnosed with IPMNs and other pancreatic cysts and those at increased genetic risk for these lesions.

In the field of pathology it is clear that molecular genomics and digital imaging represent two promising future directions, and both are as relevant to the tumor microenvironment as they are to the tumor itself (Beck AH et al. Sci Transl Med 3(108):108ra113-08ra113, 2011). Digital imaging, or whole slide imaging (WSI), of glass histology slides facilitates a number of value-added competencies which were not previously possible with the traditional analog review of these slides under a microscope by a pathologist. As an important tool for investigational research, digital pathology can leverage the quantification and reproducibility offered by image analysis to add value to the pathology field. This chapter will focus on the application of image analysis to investigate the tumor microenvironment and how quantitative investigation can provide deeper insight into our understanding of the tumor to tumor microenvironment relationship.