Upregulated expression of human neutrophil peptides 1, 2 and 3 (HNP 1-3) in colon cancer serum and tumours: a biomarker study

BACKGROUND: Molecular markers for localized colon tumours and for prognosis following therapy are needed. Proteomics research is currently producing numerous biomarker studies with clinical potential. We investigate the protein composition of plasma and of tumour extracts with the aim of identifying biomarkers for colon cancer. METHODS: By Surface Enhanced Laser Desorption/Ionisation – Time Of Flight / Mass spectrometry (SELDI-TOF/MS) we compare the protein profiles of colon cancer serum with serum from healthy individuals and the protein profiles of colon tumours with normal colon tissue. By size exclusion chromatography, we investigate the binding of HNP 1-3 to high mass plasma proteins. By microflow we investigate the effect of HNP 1-3 on mammalian cells. RESULTS: Human Neutrophil Peptides -1, -2 and -3 (HNP 1-3), also known as alfa-defensin-1, -2 and -3, are present in elevated concentrations in serum from colon cancer patients and in protein extracts from colon tumours. A fraction of HNP 1-3 in serum is bound to unidentified high mass plasma proteins. HNP 1-3 purified from colon tumours are lethal to mammalian cells. CONCLUSIONS: HNP 1-3 may serve as blood markers for colon cancer in combination with other diagnostic tools. We propose that HNP 1-3 are carried into the bloodstream by attaching to high mass plasma proteins in the tumour microenvironment. We discuss the effect of HNP 1-3 on tumour progression

Upregulated expression of human neutrophil peptides 1, 2 and 3 (HNP 1-3) in colon cancer serum and tumours: a biomarker study-1

<p><b>Copyright information:</b></p><p>Taken from "Upregulated expression of human neutrophil peptides 1, 2 and 3 (HNP 1-3) in colon cancer serum and tumours: a biomarker study"</p><p>BMC Cancer 2005;5():8-8.</p><p>Published online 19 Jan 2005</p><p>PMCID:PMC548152.</p><p>Copyright © 2005 Albrethsen et al; licensee BioMed Central Ltd.</p>0 chips. Differences in mean intensities of HNP 1-3 in normal and colon tumor tissue were statistical significant at 5% level (p < 0.0005). FIG. 2B HNP profiles of normal and colon cancer serum. Serum samples (125 colon cancer and 100 normal) were analyzed on IMAC30 chips. The mean intensities were significantly different at 5% level (p < 2.2e-16). The reproducibility was significant for all three peptides in both tissue and serum. The standard deviation of HNP 1, 2 and 3 was 70%, 136% and 57% in normal tissue and 11%, 15% and 8% in tumor tissue. The standard deviation of HNP 1, 2 and 3 was 96%, 154% and 81% in normal serum and 234%, 365% and 282% in cancer serum. The boxplot show the 25th quantile, median, 75th quantile, and whiskers extent to min. and max. values

Predicting Kinase Activity in Angiotensin Receptor Phosphoproteomes Based on Sequence-Motifs and Interactions

<div><p>Recent progress in the understanding of seven-transmembrane receptor (7TMR) signalling has promoted the development of a new generation of pathway selective ligands. The angiotensin II type I receptor (AT_1aR) is one of the most studied 7TMRs with respect to selective activation of the β-arrestin dependent signalling. Two complimentary global phosphoproteomics studies have analyzed the complex signalling induced by the AT_1aR. Here we integrate the data sets from these studies and perform a joint analysis using a novel method for prediction of differential kinase activity from phosphoproteomics data. The method builds upon NetworKIN, which applies sophisticated linear motif analysis in combination with contextual network modelling to predict kinase-substrate associations with high accuracy and sensitivity. These predictions form the basis for subsequently nonparametric statistical analysis to identify likely activated kinases. This suggested that AT_1aR-dependent signalling activates 48 of the 285 kinases detected in HEK293 cells. Of these, Aurora B, CLK3 and PKG1 have not previously been described in the pathway whereas others, such as PKA, PKB and PKC, are well known. In summary, we have developed a new method for kinase-centric analysis of phosphoproteomes to pinpoint differential kinase activity in large-scale data sets.</p></div

The angiotensin II signalling pathway.

<p>The angiotensin II signalling pathway can signal through G-protein dependent and G-protein independent pathways. The angiotensin receptor (AT_1aR) is phosphorylated upon stimulation and G-proteins activated, which leads to an increase in IP3, intracellular calcium and diacyglycerol, which activates the signalling cascade involving several kinases e.g. PKC, MAPK and PKD. Phosphorylation of the receptor also leads to the recruitment of G-protein coupled receptor kinases and β-arrestin, and ultimately to internalisation of the receptor. Internalisation of the receptor yields the second wave of signalling, and has been associated with some of the positive effects of angiotensin II signalling in the cardiac setting.</p

NetworKIN predicted active kinases.

<p>Phosphorylation sites from the two proteomic studies were analysed using the NetworKIN algorithm and the predictions were filtered based on kinases detected in the AT_1aR-HEK kinome. NetworKIN verified the activity of many of the kinases found by phospho-antibody array by Xiao et al., 2010 and predicted activity for several other kinases, of which around half have been described in the literature of angiotensin II signalling. Numbering of amino acids is according to SwissProt. Amino acids marked * are involved in the regulation of the kinase (SwissProt). Kinases that have not previously been described to be regulated in the angiotensin II signaling pathway are depicted in bold. Phosphorylation sites detected in either of the two proteomic studies Christensen et al., 2010 and Xiao et al., 2010 are denoted Ref. 4 and 5, respectively.</p

Kinases involved in the angiotensin signalling pathway.

<p>A comparison of the kinases phosphorylated in Christensen et al., 2010 and Xiao et al., 2010, with an overlay of the NetworKIN predicted active kinases. More than 30 of the kinases were differentially regulated on a phosphosite involved in kinase activity (indicated with *).</p

Comparison of the up-regulated proteins and phosphosites detected in the two MS studies.

<p>The IPI IDs of the dataset from Xiao and co-workers was mapped to UniProt, and the two ID lists were compared by an in-house script. A) Comparison of the two SII Ang II regulated data sets revealed surprisingly little overlap in phosphosites, only 5%. On the protein level the overlap was merely 17%. The figure also shows that 15 phosphosites detected to be regulated only with Ang II in the Christensen study was regulated with SII Ang II in the study by Xiao and co-workers. Of these 15 phosphosites, 5 were more than 1.4 fold regulated, and was included as SII Ang II regulated in the analysis. B) The distribution of Ser, Tyr and Thr sites in the two studies.</p

Distribution of phosphosites.

<p>The percentage of proteins with multiple phosphosites in the angiotensin phosphoproteome is 57% and the number of proteins with multiple phosphosites decreases with increasing number of regulated phosphosites. The same distribution is observed when analysing the SII Ang II regulated subset.</p