Summary The histone acetyltransferase (HAT) p300/CBP is a transcriptional coactivator implicated in many gene regulatory pathways and protein acetylation events. While p300 inhibitors have been reported, a potent, selective, and readily available active-site directed small molecule inhibitor is not yet known. Here we use a structure-based, in silico screening approach to identify a commercially available pyrazolone-containing small molecule p300 HAT inhibitor, C646. C646 is a competitive p300 inhibitor with a Ki of 400 nM and is selective versus other acetyltransferases. Studies on site-directed p300 HAT mutants and synthetic modifications of C646 confirm the importance of predicted interactions in conferring potency. Inhibition of histone acetylation and cell growth by C646 in cells validate its utility as a pharmacologic probe and suggest that p300/CBP HAT is a worthy anti-cancer target.

Protein Ser/Thr kinase CK2 (casein kinase II) is involved in a myriad of cellular processes including cell growth and proliferation by phosphorylating hundreds of substrates, yet the regulation process of CK2 function is poorly understood. Here we report that the CK2 catalytic subunit CK2α is modified by O-GlcNAc on Ser347, proximal to a cyclin-dependent kinase phosphorylation site (Thr344) on the same protein. We use protein semisynthesis to show that Thr344 phosphorylation increases CK2α cellular stability via Pin1 interaction whereas Ser347 glycosylation appears to be antagonistic to Thr344 phosphorylation and permissive to proteasomal degradation. By performing kinase assays with the site-specifically modified phospho- and glyco-modified CK2α in combination with CK2β and Pin1 binding partners on human protein microarrays, we show that CK2 kinase substrate selectivity is modulated by these specific posttranslational modifications. This study suggests how a promiscuous protein kinase can be regulated at multiple levels to achieve particular biological outputs.

Epigenetic events, including covalent post-translational modifications of histones, have been demonstrated to play critical roles in tumor development and progression. The transcriptional coactivator p300/CBP possesses both histone acetyltransferase (HAT) activity as well as scaffolding properties that directly influence the transcriptional activation of targeted genes. We have used a potent and specific inhibitor of p300/CBP HAT activity, C646, in order to evaluate the functional contributions of p300/CBP HAT to tumor development and progression. Here we report that C646 inhibits the growth of human melanoma and other tumor cells and promotes cellular senescence. Global assessment of the p300 HAT transcriptome in human melanoma identified functional roles in promoting cell cycle progression, chromatin assembly and activation of DNA repair pathways through direct transcriptional regulatory mechanisms. Additionally, C646 is shown to promote sensitivity to DNA damaging agents, leading to the enhanced apoptosis of melanoma cells following combination treatment with cisplatin. Together, our data suggest that p300 HAT activity mediates critical growth regulatory pathways in tumor cells and may serve as a potential therapeutic target for melanoma and other malignancies by promoting cellular responses to DNA damaging agents that are currently ineffective against specific cancers.

We report the feasibility of a novel, noninvasive, nonprostate specific antigen based molecular approach to detect prostate cancer in voided urine. To our knowledge this is the first report of AMACR protein detection in the urine of patients with prostate cancer. A screening test based on urinary AMACR may develop into a useful adjunct to serum prostate specific antigen and digital rectal examination for identifying men at increased risk for harboring prostate cancer despite negative biopsy. Such a test has potential application for stratifying patients into low and high risk groups for surveillance vs repeat biopsy.

Purpose: Preoperative histologic classification of solid renal masses remains limited with current technology. We determine the utility of molecular profiling based on quantitative methylation analysis for characterization of sporadic renal cell carcinoma.Experimental Design: Primary renal cell carcinomas representing three different histologic subtypes were obtained from a total of 38 patients who underwent radical nephrectomy for suspected malignant disease. Genomic DNA was isolated from tumors and was subjected to sodium bisulfite modification. The normalized index of methylation (NIM) for each sample was determined by quantitative realtime methylation-specific PCR at 17 different gene promoters. Hierarchical cluster analysis was performed by using an unsupervised neural network with binary tree topology.Results: The majority of gene promoters that were analyzed in this study demonstrated very low levels of methylation (NIM <1.0). The RASSF1A gene promoter, however, was methylated in 30 of 38 (79%) cases. The frequency of RASSF1A methylation in papillary, clear-cell, and oncocytoma subtypes was 100, 90, and 25%, respectively. The highest levels of RASSF1A methylation were observed in the papillary (mean NIM ‫؍‬ 78.9) and clear-cell (mean NIM ‫؍‬ 13.4) subtypes. The vast majority of oncocytomas were completely unmethylated, and none demonstrated >1% methylation (mean NIM ‫؍‬ 0.11). Hierarchical cluster analysis based on quantitative methylation levels resulted in stratification of sporadic renal cell carcinomas into their discrete histologic subtypes.Conclusions: Classification of sporadic renal cell carcinomas into histologic subtypes can be accomplished via multigene quantitative methylation profiling. Validation of this approach and selection of appropriate methylation markers may ultimately lead to use of this technology in the preoperative assessment of suspicious renal masses.

Gene analysis using conventional methylation specific polymerase chain reaction is a reliable method for detecting abnormal DNA methylation in voided urine samples obtained following digital rectal examination or prostate needle biopsy. The concordance between post-digital rectal examination and post-biopsy urinary samples for promoter methylation is high (82% to 94%), suggesting that urine collected after digital rectal examination may be used for genetic analysis with results similar to those in post-biopsy urine samples.

Cancer cell vascular invasion is a crucial step in the malignant progression towards metastasis. Here we used a genome-wide RNAi screen with E0771 mammary cancer cells to uncover drivers of endothelial monolayer invasion. We identified keratin-associated protein 5-5 (Krtap5-5) as a candidate. Krtap5-5 belongs to a large protein family that is implicated in crosslinking keratin intermediate filaments during hair formation, yet these keratin-associated proteins have no reported role in cancer. Depletion of Krtap5-5 from cancer cells led to cell blebbing and a loss of keratins 14 and 18, in addition to the upregulation of vimentin intermediate filaments. This intermediate filament subtype switching induced dysregulation of the actin cytoskeleton and reduced the expression of hemidesmosomal α6/β4-integrins. We further demonstrate that knockdown of keratin 18 phenocopies the loss of Krtap5-5, suggesting that Krtap5-5 crosstalks with keratin 18 in E0771 cells. Disruption of the keratin cytoskeleton by perturbing Krtap5-5 function broadly altered the expression of cytoskeleton regulators and the localization of cell surface markers. Krtap5-5 depletion did not impact cell viability but reduced cell motility and extracellular matrix invasion, as well as extravasation of cancer cells into tissues in zebrafish and mice. We conclude that Krtap5-5 is a previously unknown regulator of cytoskeletal function in cancer cells that modulates motility and vascular invasion. Thus, in addition to its physiologic function, a keratin-associated protein can serve as a switch towards malignant progression.

DNA microarray analyses were used to investigate the effect of cell-incorporated 35S-methionine on human colorectal carcinoma cells. This beta-radiation-induced gene expression profile was compared with that induced by external gamma-radiation. The extent of DNA fragmentation was used as a biomarker to determine the external gamma dose that was bioequivalent to that received by cells incubated in medium containing 35S-methionine. Studies showed that 35S-methionine at 100 microCi/mL induced a much more robust transcriptional response than gamma-radiation (2000 cGy) when evaluated 2 h after the labeling or irradiation period. The cellular response to internal beta-radiation was greater not only with respect to the number of genes induced, but also with respect to the level of gene induction. Not surprisingly, the induced genes overlapped with the set of gamma-responsive genes. However, a distinct beta-gene induction profile that included a large number of cell adhesion proteins was also observed. Taken together, these studies demonstrate that metabolic incorporation of a low energy beta-emitter, such as 35S-methionine, can globally influence a diverse set of cellular activities that can, in turn, affect the outcome of many experiments by altering the cell cycle, metabolic, signaling, or redox status (set point) of the cell. Additional studies of the mechanism of beta-induced proliferation arrest and cell death and of the significance of its differential gene induction/repression profile in comparison to pulsed gamma-irradiation may lead to new insights into the ways in which ionizing radiation can interact with cells.