An efficient procedure for protein extraction from formalin-fixed, paraffin-embedded tissues for reverse phase protein arrays

INTRODUCTION: Protein extraction from formalin-fixed paraffin-embedded (FFPE) tissues is challenging due to extensive molecular crosslinking that occurs upon formalin fixation. Reverse-phase protein array (RPPA) is a high-throughput technology, which can detect changes in protein levels and protein functionality in numerous tissue and cell sources. It has been used to evaluate protein expression mainly in frozen preparations or FFPE-based studies of limited scope. Reproducibility and reliability of the technique in FFPE samples has not yet been demonstrated extensively. We developed and optimized an efficient and reproducible procedure for extraction of proteins from FFPE cells and xenografts, and then applied the method to FFPE patient tissues and evaluated its performance on RPPA. RESULTS: Fresh frozen and FFPE preparations from cell lines, xenografts and breast cancer and renal tissues were included in the study. Serial FFPE cell or xenograft sections were deparaffinized and extracted by six different protein extraction protocols. The yield and level of protein degradation were evaluated by SDS-PAGE and Western Blots. The most efficient protocol was used to prepare protein lysates from breast cancer and renal tissues, which were subsequently subjected to RPPA. Reproducibility was evaluated and Spearman correlation was calculated between matching fresh frozen and FFPE samples. The most effective approach from six protein extraction protocols tested enabled efficient extraction of immunoreactive protein from cell line, breast cancer and renal tissue sample sets. 85% of the total of 169 markers tested on RPPA demonstrated significant correlation between FFPE and frozen preparations (p < 0.05) in at least one cell or tissue type, with only 23 markers common in all three sample sets. In addition, FFPE preparations yielded biologically meaningful observations related to pathway signaling status in cell lines, and classification of renal tissues. CONCLUSIONS: With optimized protein extraction methods, FFPE tissues can be a valuable source in generating reproducible and biologically relevant proteomic profiles using RPPA, with specific marker performance varying according to tissue type

Selenium toxicity but not deficient or super-nutritional selenium status vastly alters the transcriptome in rodents

<p>Abstract</p> <p>Background</p> <p>Protein and mRNA levels for several selenoproteins, such as glutathione peroxidase-1 (Gpx1), are down-regulated dramatically by selenium (Se) deficiency. These levels in rats increase sigmoidally with increasing dietary Se and reach defined plateaus at the Se requirement, making them sensitive biomarkers for Se deficiency. These levels, however, do not further increase with super-nutritional or toxic Se status, making them ineffective for detection of high Se status. Biomarkers for high Se status are needed as super-nutritional Se intakes are associated with beneficial as well as adverse health outcomes. To characterize Se regulation of the transcriptome, we conducted 3 microarray experiments in weanling mice and rats fed Se-deficient diets supplemented with up to 5 μg Se/g diet.</p> <p>Results</p> <p>There was no effect of Se status on growth of mice fed 0 to 0.2 μg Se/g diet or rats fed 0 to 2 μg Se/g diet, but rats fed 5 μg Se/g diet showed a 23% decrease in growth and elevated plasma alanine aminotransferase activity, indicating Se toxicity. Rats fed 5 μg Se/g diet had significantly altered expression of 1193 liver transcripts, whereas mice or rats fed ≤ 2 μg Se/g diet had < 10 transcripts significantly altered relative to Se-adequate animals within an experiment. Functional analysis of genes altered by Se toxicity showed enrichment in cell movement/morphogenesis, extracellular matrix, and development/angiogenesis processes. Genes up-regulated by Se deficiency were targets of the stress response transcription factor, Nrf2. Multiple regression analysis of transcripts significantly altered by 2 μg Se/g and Se-deficient diets identified an 11-transcript biomarker panel that accounted for 99% of the variation in liver Se concentration over the full range from 0 to 5 μg Se/g diet.</p> <p>Conclusion</p> <p>This study shows that Se toxicity (5 μg Se/g diet) in rats vastly alters the liver transcriptome whereas Se-deficiency or high but non-toxic Se intake elicits relatively few changes. This is the first evidence that a vastly expanded number of transcriptional changes itself can be a biomarker of Se toxicity, and that identified transcripts can be used to develop molecular biomarker panels that accurately predict super-nutritional and toxic Se status.</p

Looking at Cerebellar Malformations through Text-Mined Interactomes of Mice and Humans

WE HAVE GENERATED AND MADE PUBLICLY AVAILABLE TWO VERY LARGE NETWORKS OF MOLECULAR INTERACTIONS: 49,493 mouse-specific and 52,518 human-specific interactions. These networks were generated through automated analysis of 368,331 full-text research articles and 8,039,972 article abstracts from the PubMed database, using the GeneWays system. Our networks cover a wide spectrum of molecular interactions, such as bind, phosphorylate, glycosylate, and activate; 207 of these interaction types occur more than 1,000 times in our unfiltered, multi-species data set. Because mouse and human genes are linked through an orthological relationship, human and mouse networks are amenable to straightforward, joint computational analysis. Using our newly generated networks and known associations between mouse genes and cerebellar malformation phenotypes, we predicted a number of new associations between genes and five cerebellar phenotypes (small cerebellum, absent cerebellum, cerebellar degeneration, abnormal foliation, and abnormal vermis). Using a battery of statistical tests, we showed that genes that are associated with cerebellar phenotypes tend to form compact network clusters. Further, we observed that cerebellar malformation phenotypes tend to be associated with highly connected genes. This tendency was stronger for developmental phenotypes and weaker for cerebellar degeneration

Integrative Network Biology: Graph Prototyping for Co-Expression Cancer Networks

Network-based analysis has been proven useful in biologically-oriented areas, e.g., to explore the dynamics and complexity of biological networks. Investigating a set of networks allows deriving general knowledge about the underlying topological and functional properties. The integrative analysis of networks typically combines networks from different studies that investigate the same or similar research questions. In order to perform an integrative analysis it is often necessary to compare the properties of matching edges across the data set. This identification of common edges is often burdensome and computational intensive. Here, we present an approach that is different from inferring a new network based on common features. Instead, we select one network as a graph prototype, which then represents a set of comparable network objects, as it has the least average distance to all other networks in the same set. We demonstrate the usefulness of the graph prototyping approach on a set of prostate cancer networks and a set of corresponding benign networks. We further show that the distances within the cancer group and the benign group are statistically different depending on the utilized distance measure

Gene Expression and Biological Pathways in Tissue of Men with Prostate Cancer in a Randomized Clinical Trial of Lycopene and Fish Oil Supplementation

Studies suggest that micronutrients may modify the risk or delay progression of prostate cancer; however, the molecular mechanisms involved are poorly understood. We examined the effects of lycopene and fish oil on prostate gene expression in a double-blind placebo-controlled randomized clinical trial.Eighty-four men with low risk prostate cancer were stratified based on self-reported dietary consumption of fish and tomatoes and then randomly assigned to a 3-month intervention of lycopene (n = 29) or fish oil (n = 27) supplementation or placebo (n = 28). Gene expression in morphologically normal prostate tissue was studied at baseline and at 3 months via cDNA microarray analysis. Differential gene expression and pathway analyses were performed to identify genes and pathways modulated by these micronutrients.Global gene expression analysis revealed no significant individual genes that were associated with high intake of fish or tomato at baseline or after 3 months of supplementation with lycopene or fish oil. However, exploratory pathway analyses of rank-ordered genes (based on p-values not corrected for multiple comparisons) revealed the modulation of androgen and estrogen metabolism in men who routinely consumed more fish (p = 0.029) and tomato (p = 0.008) compared to men who ate less. In addition, modulation of arachidonic acid metabolism (p = 0.01) was observed after 3 months of fish oil supplementation compared with the placebo group; and modulation of nuclear factor (erythroid derived-2) factor 2 or Nrf2-mediated oxidative stress response for either supplement versus placebo (fish oil: p = 0.01, lycopene: p = 0.001).We did not detect significant individual genes associated with dietary intake and supplementation of lycopene and fish oil. However, exploratory analyses revealed candidate in vivo pathways that may be modulated by these micronutrients.ClinicalTrials.gov NCT00402285

The role of Sprouty-dependent inhibition of the Ras /ERK pathway in melanoma cells: A systems biology approach

Ras/ERK pathway signaling is frequently altered in melanoma. The discovery of mutations in the BRAF gene that lead to activation of the pathway suggested BRAF as a potential target for melanoma therapy. In order to design and optimize future therapeutic strategies for melanoma, it is important to identify additional factors that modulate the function of BRAF or cause activation of Ras/ERK signaling in the absence of a BRAF mutation. In order to identify and evaluate such factors, we used systems biology methodologies that enable the study of signaling networks with multiple components and interconnections. Towards this end, microarray-based gene expression profiling was performed for normal melanocytes and melanoma cell lines with or without a BRAF mutation. Data analysis of the Ras/ERK gene expression patterns revealed that the expression of the inhibitor SPRY2 was reduced in the cells without a BRAF mutation. This observation suggested that reduction in SPRY2 expression may be an alternative mechanism of activating the pathway in the absence of BRAF mutant. Subsequent functional analysis of SPRY2 using siRNA-mediated knockdown approaches in melanoma cells showed that upon SPRY2 knock-down by siRNA, ERK signaling is increased, but only in wild-type BRAF cells. The data suggested that SPRY2 is an inhibitor of the Ras/ERK pathway, but only when the BRAF mutation is absent. A potential mechanism of SPRY2 inhibition involves direct binding of SPRY2 to BRAF, as demonstrated by co-immunoprecipitation experiments. This interaction is disrupted by the presence of mutations in the kinase domain of BRAF, providing a potential explanation for the absence of SPRY2-dependent inhibition in the presence of BRAF mutations. The results of the functional analysis suggested that SPRY2 may play a role as a tumor suppressor being down-regulated in melanoma of wild-type BRAF status, and it is therefore of interest to understand its expression regulation. In this study, we constructed a computational model, which was based upon information provided by the functional analysis of SPRY2 mentioned above. The model was then used to explore SPRY2 expression regulation, by studying its responsiveness to an ERK-induced feedback loop. The computational model proposed that the Elk1-phosphatase PP2B modulates the ERK-induced regulation of SPRY2 expression, and predicted down-regulation of PP2B in a melanoma cell line. The model-driven hypotheses were then confirmed with biological experiments. The findings suggest that the behavior of the Ras/ERK pathway depends on alterations such as the BRAF mutation, differential SPRY2 levels and PP2B. Each of these pathway components may vary across different melanoma tumors, giving rise to complex pathway behaviors that can be evaluated by high-throughput profiling and computational approaches

The role of Sprouty-dependent inhibition of the Ras /ERK pathway in melanoma cells: A systems biology approach

Ras/ERK pathway signaling is frequently altered in melanoma. The discovery of mutations in the BRAF gene that lead to activation of the pathway suggested BRAF as a potential target for melanoma therapy. In order to design and optimize future therapeutic strategies for melanoma, it is important to identify additional factors that modulate the function of BRAF or cause activation of Ras/ERK signaling in the absence of a BRAF mutation. In order to identify and evaluate such factors, we used systems biology methodologies that enable the study of signaling networks with multiple components and interconnections. Towards this end, microarray-based gene expression profiling was performed for normal melanocytes and melanoma cell lines with or without a BRAF mutation. Data analysis of the Ras/ERK gene expression patterns revealed that the expression of the inhibitor SPRY2 was reduced in the cells without a BRAF mutation. This observation suggested that reduction in SPRY2 expression may be an alternative mechanism of activating the pathway in the absence of BRAF mutant. Subsequent functional analysis of SPRY2 using siRNA-mediated knockdown approaches in melanoma cells showed that upon SPRY2 knock-down by siRNA, ERK signaling is increased, but only in wild-type BRAF cells. The data suggested that SPRY2 is an inhibitor of the Ras/ERK pathway, but only when the BRAF mutation is absent. A potential mechanism of SPRY2 inhibition involves direct binding of SPRY2 to BRAF, as demonstrated by co-immunoprecipitation experiments. This interaction is disrupted by the presence of mutations in the kinase domain of BRAF, providing a potential explanation for the absence of SPRY2-dependent inhibition in the presence of BRAF mutations. The results of the functional analysis suggested that SPRY2 may play a role as a tumor suppressor being down-regulated in melanoma of wild-type BRAF status, and it is therefore of interest to understand its expression regulation. In this study, we constructed a computational model, which was based upon information provided by the functional analysis of SPRY2 mentioned above. The model was then used to explore SPRY2 expression regulation, by studying its responsiveness to an ERK-induced feedback loop. The computational model proposed that the Elk1-phosphatase PP2B modulates the ERK-induced regulation of SPRY2 expression, and predicted down-regulation of PP2B in a melanoma cell line. The model-driven hypotheses were then confirmed with biological experiments. The findings suggest that the behavior of the Ras/ERK pathway depends on alterations such as the BRAF mutation, differential SPRY2 levels and PP2B. Each of these pathway components may vary across different melanoma tumors, giving rise to complex pathway behaviors that can be evaluated by high-throughput profiling and computational approaches