Quantitative Proteomics Identifies Activation of Hallmark Pathways of Cancer in Patient Melanoma.

Molecular pathways regulating melanoma initiation and progression are potential targets of therapeutic development for this aggressive cancer. Identification and molecular analysis of these pathways in patients has been primarily restricted to targeted studies on individual proteins. Here, we report the most comprehensive analysis of formalin-fixed paraffin-embedded human melanoma tissues using quantitative proteomics. From 61 patient samples, we identified 171 proteins varying in abundance among benign nevi, primary melanoma, and metastatic melanoma. Seventy-three percent of these proteins were validated by immunohistochemistry staining of malignant melanoma tissues from the Human Protein Atlas database. Our results reveal that molecular pathways involved with tumor cell proliferation, motility, and apoptosis are mis-regulated in melanoma. These data provide the most comprehensive proteome resource on patient melanoma and reveal insight into the molecular mechanisms driving melanoma progression.

Misregulation of Rad50 expression in melanoma cells.

DNA double-strand breaks are increased in human melanoma tissue as detected by histone H2AX phosphorylation.(1-3) We investigated two of the downstream effectors of DNA double-strand breaks, Rad50 and 53BP1 (tumor suppressor p53 binding protein 1), to determine if they are altered in human primary melanoma cells. Melanoma cases showed high Rad50 staining (81.8%; 9/11) significantly more frequently than conventional or atypical melanocytic nevi (0%; 0/18). In contrast, the staining pattern for 53BP1 appears similar between melanoma and nevi. This is the first study that shows activation and misregulation of the DNA repair pathway in human melanoma cells. The staining features of Rad50, a component of an essential DNA double-strand break repair complex, are clearly increased in melanoma cells with regards to both staining intensity and the number of positive melanoma cells. Interestingly, among the melanoma cases with increased Rad50 staining, most demonstrated cytoplasmic rather than nuclear staining (88.9%, 8/9). Further studies are needed to determine the cause of this mislocalization and its affects, if any, on DNA double-strand break repair in melanoma.

Analysis of stable and transient protein-protein interactions.

The assembly of proteins into defined complexes drives a plethora of cellular activities. These protein complexes often have a set of more stably interacting proteins as well as more unstable or transient interactions. Studying the in vivo components of these protein complexes is challenging as many of the techniques used for isolation result in the purification of only the most stable components and the transient interactions are lost. A technology called transient isotopic differentiation of interactions as random or targeted (transient I-DIRT) has been developed to identify these transiently interacting proteins as well as the stable interactions. Described here are the detailed methodological approaches used for a transient I-DIRT analysis of a multi-subunit complex, NuA3, that acetylates histone H3 and functions to activate gene transcription. Transcription is known to involve a concert of protein assemblies performing different activities on the chromatin/gene template, thus understanding the less stable or transient protein interactions with NuA3 will shed light onto the protein complexes that function synergistically, or antagonistically, to regulate gene transcription and chromatin remodeling.

A noncanonical bromodomain in the AAA ATPase protein Yta7 directs chromosomal positioning and barrier chromatin activity.

Saccharomyces cerevisiae Yta7 is a barrier active protein that modulates transcriptional states at the silent mating locus, HMR. Additionally, Yta7 regulates histone gene transcription and has overlapping functions with known histone chaperones. This study focused on deciphering the functional role of the noncanonical Yta7 bromodomain. By use of genetic and epistasis analyses, the Yta7 bromodomain was shown to be necessary for barrier activity at HMR and to have overlapping functions with histone regulators (Asf1 and Spt16). Canonical bromodomains can bind to acetylated lysines on histones; however, the Yta7 bromodomain showed an association with histones that was independent of posttranslational modification. Further investigation showed that regions of Yta7 other than the bromodomain conferred histone association. Chromatin immunoprecipitation-chip analyses revealed that the Yta7 bromodomain was not solely responsible for histone association but was also necessary for proper chromosomal positioning of Yta7. This work demonstrates that the Yta7 bromodomain engages histones for certain cellular functions like barrier chromatin maintenance and particular Spt16/Asf1 cellular pathways of chromatin regulation.

Environmental exposures and puberty in inner-city girls.

BACKGROUND: Hormonally active environmental exposures are suspected to alter onset of puberty in girls, but research on this question has been very limited. OBJECTIVE: We investigated pubertal status in relation to hormonally active environmental exposures among a multiethnic group of 192 healthy 9-year-old girls residing in New York City. METHODS: Information was collected on breast and pubic hair stages, weight and height. Phytoestrogen intake was estimated from a food-frequency questionnaire. Three phytoestrogens and bis-phenolA (BPA) were measured in urine. In a subset, 1,1'-dichloro-2,2'-bis(4-chlorophenyl)ethylene (DDE), polychlorinated biphenyls (PCBs) were measured in blood plasma and lead (Pb) in blood. Associations of exposures with pubertal stages (present=stage 2+ vs absent=stage 1) were examined using t-tests and Poisson multivariate regression to derive prevalence ratios (PR, 95%-confidence limits [CI]). RESULTS: Breast development was present in 53% of girls. DDE, Pb, and dietary intakes of phytoestrogens were not significantly associated with breast stage. Urinary phytoestrogen biomarker concentrations were lower among girls with breast development compared with no development. In multivariate models, main effects were strongest for two urinary isoflavones, daidzein (PR 0.89 [0.83-0.96] per ln microg/g creatinine) and genistein (0.94 [0.88-1.01]). Body mass index (BMI) is a hormonally relevant, strong risk factor for breast development. Therefore, BMI-modification of exposure effects was examined, and associations became stronger. Delayed breast development was observed among girls with below-median BMI and third tertile (high exposure) of urinary daidzein (PR 0.46 [0.26-0.78]); a similar effect was seen with genistein, comparing to girls >or= median BMI and lowest two tertiles (combined) of these isoflavones. With urinary enterolactone a phytoestrogen effect was seen only among girls with high BMI, where breast development was delayed among those with high urinary enterolactone (PR 0.55 [0.32-0.96] for the upper tertile vs lower two combined). There was no main effect of PCBs on breast stage, but girls with below-median BMI and >or= median PCB levels had reduced risk for breast development (any vs none) compared with other BMI-PCB groups. No biomarkers were associated with hair development, which was present in 31% of girls. CONCLUSIONS: Phytoestrogens and PCBs are environmental exposures that may delay breast development, especially in conjunction with BMI, which governs the endogenous hormonal milieu. Further research to confirm these findings may improve our understanding of the role of early life development in breast cancer risk and other chronic diseases related to obesity.

Characteristics of pubertal development in a multi-ethnic population of nine-year-old girls.

PURPOSE: Early age at menarche increases future disease risk. Secular decline in age at menarche has been attributed to body size characteristics, diet, and energy expenditure. Risk factors for puberty have been less frequently explored. METHODS: A cross-sectional study of 186 New York Metropolitan Area, 9-year-old girls (54 African-American, 70 Hispanic, 62 Caucasians) used interviewer-administered questionnaires to assess exposures. Height and weight were measured. Pediatricians assessed pubertal development according to Tanner stages. RESULTS: African-Americans were more likely than Caucasians to have achieved puberty as determined by breast or hair development (stage 2 or higher) [age-adjusted odds ratios and 95% confidence intervals = 4.91 (2.15-11.19) and 4.25 (1.85-9.77), respectively]. Pubertal development was similar among Hispanics and Caucasians. Adiposity and height were significantly positively associated with breast or hair development. More sedentary activity hours non-significantly increased the likelihood of hair development. Lower energy, but higher polyunsaturated fat, consumption were suggestive of an association with breast development. Vitamin C and hair development were inversely related. No other nutrients or physical activity measures were related to pubertal development. CONCLUSIONS: Results are consistent with height and adiposity being associated with pubertal development. Sedentary activity or diet might possibly influence maturation.