Identification of Clinically Relevant Protein Targets in Prostate Cancer with 2D-DIGE Coupled Mass Spectrometry and Systems Biology Network Platform

Prostate cancer (PCa) is the most common type of cancer found in men and among the leading causes of cancer death in the western world. In the present study, we compared the individual protein expression patterns from histologically characterized PCa and the surrounding benign tissue obtained by manual micro dissection using highly sensitive two-dimensional differential gel electrophoresis (2D-DIGE) coupled with mass spectrometry. Proteomic data revealed 118 protein spots to be differentially expressed in cancer (n = 24) compared to benign (n = 21) prostate tissue. These spots were analysed by MALDI-TOF-MS/MS and 79 different proteins were identified. Using principal component analysis we could clearly separate tumor and normal tissue and two distinct tumor groups based on the protein expression pattern. By using a systems biology approach, we could map many of these proteins both into major pathways involved in PCa progression as well as into a group of potential diagnostic and/or prognostic markers. Due to complexity of the highly interconnected shortest pathway network, the functional sub networks revealed some of the potential candidate biomarker proteins for further validation. By using a systems biology approach, our study revealed novel proteins and molecular networks with altered expression in PCa. Further functional validation of individual proteins is ongoing and might provide new insights in PCa progression potentially leading to the design of novel diagnostic and therapeutic strategies

Effect of Hydrocortisone on Mortality and Organ Support in Patients With Severe COVID-19: The REMAP-CAP COVID-19 Corticosteroid Domain Randomized Clinical Trial.

Importance: Evidence regarding corticosteroid use for severe coronavirus disease 2019 (COVID-19) is limited. Objective: To determine whether hydrocortisone improves outcome for patients with severe COVID-19. Design, Setting, and Participants: An ongoing adaptive platform trial testing multiple interventions within multiple therapeutic domains, for example, antiviral agents, corticosteroids, or immunoglobulin. Between March 9 and June 17, 2020, 614 adult patients with suspected or confirmed COVID-19 were enrolled and randomized within at least 1 domain following admission to an intensive care unit (ICU) for respiratory or cardiovascular organ support at 121 sites in 8 countries. Of these, 403 were randomized to open-label interventions within the corticosteroid domain. The domain was halted after results from another trial were released. Follow-up ended August 12, 2020. Interventions: The corticosteroid domain randomized participants to a fixed 7-day course of intravenous hydrocortisone (50 mg or 100 mg every 6 hours) (n = 143), a shock-dependent course (50 mg every 6 hours when shock was clinically evident) (n = 152), or no hydrocortisone (n = 108). Main Outcomes and Measures: The primary end point was organ support-free days (days alive and free of ICU-based respiratory or cardiovascular support) within 21 days, where patients who died were assigned -1 day. The primary analysis was a bayesian cumulative logistic model that included all patients enrolled with severe COVID-19, adjusting for age, sex, site, region, time, assignment to interventions within other domains, and domain and intervention eligibility. Superiority was defined as the posterior probability of an odds ratio greater than 1 (threshold for trial conclusion of superiority >99%). Results: After excluding 19 participants who withdrew consent, there were 384 patients (mean age, 60 years; 29% female) randomized to the fixed-dose (n = 137), shock-dependent (n = 146), and no (n = 101) hydrocortisone groups; 379 (99%) completed the study and were included in the analysis. The mean age for the 3 groups ranged between 59.5 and 60.4 years; most patients were male (range, 70.6%-71.5%); mean body mass index ranged between 29.7 and 30.9; and patients receiving mechanical ventilation ranged between 50.0% and 63.5%. For the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively, the median organ support-free days were 0 (IQR, -1 to 15), 0 (IQR, -1 to 13), and 0 (-1 to 11) days (composed of 30%, 26%, and 33% mortality rates and 11.5, 9.5, and 6 median organ support-free days among survivors). The median adjusted odds ratio and bayesian probability of superiority were 1.43 (95% credible interval, 0.91-2.27) and 93% for fixed-dose hydrocortisone, respectively, and were 1.22 (95% credible interval, 0.76-1.94) and 80% for shock-dependent hydrocortisone compared with no hydrocortisone. Serious adverse events were reported in 4 (3%), 5 (3%), and 1 (1%) patients in the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively. Conclusions and Relevance: Among patients with severe COVID-19, treatment with a 7-day fixed-dose course of hydrocortisone or shock-dependent dosing of hydrocortisone, compared with no hydrocortisone, resulted in 93% and 80% probabilities of superiority with regard to the odds of improvement in organ support-free days within 21 days. However, the trial was stopped early and no treatment strategy met prespecified criteria for statistical superiority, precluding definitive conclusions. Trial Registration: ClinicalTrials.gov Identifier: NCT02735707

A–C: Venn diagrams for representation of drug specific protein expression.

<p>Venn diagrams illustrated the drug specific effects in different cell lines: (<b>A</b>) Ba/F3-p210, (<b>B</b>) Ba/F3-M351T and (<b>C</b>) Ba/F3-T351I cells. The numbers inside the circles represent the number of regulated proteins.</p

Schematic representation of meso network architecture and experimental design.

<p>(<b>A</b>) Exemplifies an abstract meso-scale network representing (abstract) pathways of drug action of drugs A and B on induction of proteins (red, yellow and orange bullets). Drug A uses two pathways (blue and black), whereas the blue pathway induces expression shifts only on a subset of the proteins (red, yellow), whereas the black pathway induces expression shifts in all proteins. Drug B acts only via one pathway which joins the black pathway of Drug A in an abstract node (represented by the green bullet). The mutation inhibits both pathways between the green and blue bullet resulting in an interference with drug induced expression shift for all proteins. The blue pathway from Drug A to the proteins, however, is not affected by the mutation. Hence the mutation may have a strong impact on the efficacy of drug B, whereas the profile of action of drug A is only altered by the mutation. (B) Imatinib sensitive and resistant cells were treated with four tyrosine kinase inhibitor and mesoscale network were reengineered based on specific proteome expression patterns.</p

A–E: Meso scale networks Ba/F3-p210 cells.

<p>(<b>A</b>) High degree of co-regulation across the protein set for IM, DASA and NILO, which can effectively represented by the mean component of factor analysis. (<b>B</b>) Significant deviations for a small set of proteins for DANU suggesting the use of the more stable factor analysis instead of PCA for reduction of dimension. (<b>C</b>) Analysis quantitatively the amount of induction of protein expression, which is associated with the activation of the dominant mechanisms, quantified by the mean component of factor analysis. Whereas a good and almost similar behaviour for IM, DASA and NILO is observed, DANU activates the proteins in two clearly separated modes (indicated by the upper and lower line of red stars). This finding is supported by quantitatively testing the distribution of the residuals of protein expressions with respect to the linear regression model given by the mean component of factor analysis. (<b>D</b>) Apparently only DANU induces residuals with significant non-gaussian noise indicating the existence of two separate mechanisms of protein induction. (<b>E</b>) Structure of meso scale pathways for induced protein expression. Black block represents induction of the protein expression by the main pathway, whereas the red block is indicating an inhibition via the main pathway.</p

A–D: Meso scale network models for apoptosis induction.

<p>(<b>A</b>) Distribution of the Pearson coefficient between individual protein expression and the mean component of factor analysis which represents the dominant co-regulation mechanism. (<b>B</b>) Shows that the proteins can be decomposed into two groups differing with respect to the impact of DANU. Most proteins show no different co-regulation behaviour if DANU is omitted from the data set, whereas three proteins show a significantly higher degree of co-regulation (increased r value) when DANU is omitted indicating a second mode of action of DANU. (<b>C</b>) Shows that with exception of two treatments the mean protein expression, represented by the value of the mean component of the factor analysis (y-axis) is correlated to the observed induction of apoptosis (x-axis) indicating a similar efficacy in apoptosis induction for most drugs. The exceptions indicate that protein expression is induced which does not contribute to apoptosis induction. (<b>D</b>) Depicts the meso scale set of pathways, which fits two the observations.</p

A–D: Meso scale networks Ba/F3-M351T.

<p>(<b>A</b>) Shows the protein expressions for wt and T351 cell type for all drugs. (<b>B</b>) Shows the sensitivity of protein expression with respect to the dominant activation mechanism, quantified by the mean component of factor analysis. (<b>C</b>) Shows, that surprisingly the overall level of protein expression induced by NILO increases, although the sensitivity decreases. (<b>D</b>) Shows the modifications which are induced by the analysis of the M351I mutation to the meso scale pathway network depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053668#pone-0053668-g005" target="_blank">Figure 5E</a>. Black block represents induction of the protein expression by the main pathway, whereas the red block is indicating an inhibition via the main pathway. Green block represents the unique effect of NILO on the overall protein expression level.</p

A–D: Western blot analyses revealed posttranslational modification of eIF5A and up regulation of TGM2 after treatment with IM.

<p>(<b>A</b>) Enlarged regions from a coomassie stained 2D-PAGE from Ba/F3-p210 cells after treatment with IM or DMSO as a control. The arrows indicate two spots for eIF5A, one at pI of 5.2 and the other one at a pI of 6.1. The latter appeared after IM treatment. (<b>B</b>) 2D-WB validated the appearance of a second spot for eIF5A at a pI of 6.2 after IM treatment. (<b>C</b>) Enlarged regions from a coomassie stained 2D-PAGE from Ba/F3-p210 cells after treatment with IM or DMSO as a control. One spot for TGM2 (arrow) demonstrated an increased expression after IM treatment. (<b>D</b>) The increased expression of TGM2 after treatment with rising concentrations of IM could be validated in human Bcr-Abl K562 cells.</p