Differential expression of serum clusterin isoforms in colorectal cancer

Clusterin is an enigmatic protein altered in tumors of colorectal cancer patients. Because there is no information available about serum clusterin regarding this pathology, we applied proteomic techniques to analyze its isoforms in donors and patients. First we separated serum proteins through concanavalin A, obtaining a fraction with non- and O-glycosylated proteins (FI) and a second fraction enriched in N-glycoproteins (FII) wherein clusterin was supposed to elute on the basis of its glycosylation. Surprisingly analysis of the FI fraction revealed the existence of an unexpected and aberrantly N-glycosylated clusterin that was overexpressed in patients and comprised at least five isoforms with different isoelectric points. On the other hand, two-dimensional electrophoretic analysis of the clusterin eluted in FII detected one isoform that was increased and 15 isoforms that were decreased or absent in serum of patients. Finally immunoquantification by slot blot showed that in total serum and in FI the clusterin levels were significantly increased in patients, whereas in FII there was no significant variation. Therefore, serum clusterin and some of its isoforms could have a potential value as colorectal tumor markers and are interesting subjects for biomarker studies.     

Cloning and characterization of a human GDPD domain-containing protein GDPD5

Glycerophosphodiester phosphodiesterase (GDPD) catalyzes the hydrolysis of deacylated glycerophospholipids to glycerol phosphate and alcohol. GDPD5 has been reported in Mus musculus and Gallus gallus, but not in Homo sapiens. Here we report the cloning and characterization of a novel human GDPD domain-containing gene, GDPD5, isolated from human testis cDNA library, and mapped to 11q13.4-13.5 by searching the UCSC genomic database. The GDPD5 cDNA sequence of 3442 base pairs contains an open reading frame encoding 605 amino acids. The GDPD5 gene consists of 17 exons and encodes a putative protein with six transmembrane regions and a GDPD motif. Subcellular localization of GDPD5 demonstrated that the protein was localized in the cytoplasm when overexpressed in COS-7 cells. RT-PCR analysis showed that GDPD5 was widely expressed in human tissues and the expression levels in kidney and prostate were relatively low.     

Differential protein expression on the cell surface of colorectal cancer cells associated to tumor metastasis

Progression to metastasis is the critical point in colorectal cancer (CRC) survival. However, the proteome associated to CRC metastasis is very poorly understood at the moment. In this study, we used stable isotope labeling by amino acids in cell culture to compare two CRC cell lines: KM12C and KM12SM, representing poorly versus highly metastatic potential, to find and quantify the differences in protein expression, mostly at the cell surface level. After biotinylation followed by affinity purification, membrane proteins were separated by SDS‐PAGE and analyzed using nanoflow LC‐ESI‐LTQ. A total of 291 membrane and membrane‐associated proteins were identified with a p value<0.01, from which 60 proteins were found to be differentially expressed by more than 1.5‐fold. We identified a number of cell signaling, CDs, integrins and other cell adhesion molecules (cadherin 17, junction plakoglobin (JUP)) among the most deregulated proteins. They were validated by Western blot, confocal microscopy and flow cytometry analysis. Immunohistochemical analysis of paired tumoral samples confirmed that these differentially expressed proteins were also altered in human tumoral tissues. A good correlation with a major abundance in late tumor stages was observed for JUP and 17‐β‐hydroxysteroid dehydrogenase type 8 (HSD17B8). Moreover, the combined increase in JUP, occludin and F11 receptor expression together with cadherin 17 expression could suggest a reversion to a more epithelial phenotype in highly metastatic cells. Relevant changes were observed also at the metabolic level in the pentose phosphate pathway and several amino acid transporters. In summary, the identified proteins provide us with a better understanding of the events involved in liver colonization and CRC metastasis.     

Study of P53 protein expression in colorectal cancer

Mutations of the P53 gene, strictly associated with the carcinogenesis are a commonly observed in neoplastic cells. The aim of this study was the immunohistochemical evaluation of P53 protein expression in colorectal carcinomas and analysis of its relationship to chosen anatomo-clinical and morphological parameters of the tumours. The study used the material obtained during surgical treatment of 74 colorectal carcinomas. Tissue sections were fixed in 10% buffered formaldehyde solution, embedded in paraffin and stained immunohistochemically with the antihuman P53 protein monoclonal antibody. The immunolocalization of P53 protein was performed using the Labelled Streptavidin Biotin (LSAB) method. The P53 protein expression was semiquantitatively assessed in neoplastic cells and the reaction present in more than 25% of tumour cells was accepted as the threshold of positivity. No correlation was found between P53 protein expression and tumour histologic type and site, and age and sex of patients. However, P53 protein expression in primary and metastatic tumours was found statistically significantly correlated.     

Differential expression of proteomics models of colorectal cancer, colorectal benign disease and healthy controls

Background

Colorectal cancer (CRC) is often diagnosed at a late stage with concomitant poor prognosis. The hypersensitive analytical technique of proteomics can detect molecular changes before the tumor is palpable. The surface-enhanced laser desorption/ionization-time of flight-mass spectra (SELDI-TOF-MS) is a newly-developed technique of evaluating protein separation in recent years. The protein chips have established the expression of tumor protein in the serum specimens and become the newly discovered markers for tumor diagnosis. The objective of this study was to find new markers of the diagnosis among groups of CRC, colorectal benign diseases (CBD) and healthy controls. The assay of SELDI-TOF-MS with analytical technique of protein-chip bioinformatics was used to detect the expression of protein mass peaks in the sera of patients or controls. One hundred serum samples, including 52 cases of colorectal cancer, 27 cases of colorectal benign disease, and 21 cases of healthy controls, were examined by SELDI-TOF-MS with WCX2 protein-chips.

Results

The diagnostic models (I, II and III) were setup by analyzed the data and sieved markers using Ciphergen - Protein-Chip-Software 5.1. These models were combined with 3 protein mass peaks to discriminate CRC, CBD, and healthy controls. The accuracy, the sensitivity and the particularity of cross verification of these models are all highly over 80%.

Conclusions

The SELDI-TOF-MS is a useful tool to help diagnose colorectal cancer, especially during the early stage. However, identification of the significantly differentiated proteins needs further study.     

The role of peptide deformylase in protein biosynthesis: a proteomic study

Recently we investigated the influence of classical and emerging antibiotics on the proteome of Bacillus subtilis including in our studies actinonin, a potent novel inhibitor of peptide deformylase. The protein synthesis pattern under actinonin treatment changed so dramatically that a direct comparison to the control pattern was impossible. Dual channel imaging revealed that actinonin treatment caused the majority of newly synthesised proteins to accumulate in spots different from the ones usually observed, indicating a more acidic isoelectric point. Two strategies were used to investigate the nature of the charge shift. In the first place, protein patterns of a conditional peptide deformylase mutant under nonrepressing and repressing conditions were compared. Secondly, several protein pairs excised from two-dimensional (2-D) gels of the peptide deformylase mutant, exponentially growing untreated wild-type and the actinonin treated wild-type were investigated with matrix-assisted laser desorption/ionization and electrospray ionization (ESI) time of flight mass spectrometry (TOF MS) for the existence of N-terminal formylation. Under nonrepressing conditions the mutant protein pattern resembled that of the wild-type. The loss of peptide deformylase activity under repressing conditions led to the same pI shift observed for actinonin treatment in the wild-type. Quadrupole TOF-MS on 11 protein pairs proved that the remaining N-terminal formyl residue was indeed responsible for the charge shift. Eight of these protein pairs were also present on 2-D gels of exponentially growing B. subtilis, where the more acidic, still formylated protein species represented the smaller parts.     

Identification of multi-SH3 domain-containing protein interactome in pancreatic cancer: a yeast two-hybrid approach

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease that shows minimal response to chemotherapy. Genetic changes involved in the progression of PDAC concern genes that encode proteins related to signal transduction networks. This fact reveals the importance in identifying the role and the relations between multiple signaling cascades in PDAC. One of the major factors that modulate signaling events is multidomain scaffold proteins that function by binding several proteins simultaneously, inducing their proximity and influencing the outcome of signaling. A particular group among them, containing multiple Src homology 3 (SH3) domains that can bind proteins containing proline-rich motifs, was associated to different aspects of cancer cell homeostasis. In this work, using a microarray-based analysis, we have shown that 13 multiple SH3 domain containing scaffold proteins are expressed in PDAC cells. Using a yeast two-hybrid approach, we have identified proteins that interact with these adaptor proteins. Among them we have found several molecules that modulate cell proliferation and survival (CIZ1, BIRC6, RBBP6), signaling (LTBP4, Notch2, TOM1L1, STK24) and membrane dynamics (PLSCR1, DDEF2, VCP). Our results indicate that interactions mediated by multi-SH3 domain-containing proteins could lead to the formation of dynamic protein complexes that function in pancreatic cancer cell signaling. The identification of such protein complexes is of paramount importance in deciphering pancreatic cancer biology and designing novel therapeutic approaches.     

Genetic variability in IL23R and risk of colorectal adenoma and colorectal cancer

Inflammatory processes, including, specifically, the inflammatory conditions Crohn's disease (CD) and ulcerative colitis (UC) predispose to colorectal cancer. Interleukin-23 is involved in pro-inflammatory signaling; genetic variation in the interleukin-23 receptor (IL23R) has been consistently associated with CD and UC risk. In three case-control studies of colorectal adenoma (n=485 cases/578 controls), colon cancer (n=1424 cases/1780 controls) and rectal cancer (n=583 cases/775 controls), we investigated associations with 18 candidate and tagSNPs in IL23R. The three studies used an identical Illumina GoldenGate assay, allowing thorough investigation across stages and locations of colorectal neoplasia. We further explored associations with molecular cancer subtypes (MSI+, CIMP+, KRAS2mut, TP53mut). In this comprehensive study of genetic variability in IL23R across the spectrum of colorectal carcinogenesis, as well as within colon and rectal tumor molecular subtypes, we observed associations between SNPs in IL23R and risk of rectal cancer: the 88413 C>A (rs10889675) and 69450 C>A (rs7542081) polymorphisms were associated with decreased rectal cancer risk overall (p-trend=0.04 and 0.05 respectively), and specifically with rectal tumors bearing a TP53 mutation (88413 CA/AA vs. CC OR: 0.66; 95% CI: 0.46-94; 69450 CA/AA vs. CC OR: 0.60; 95% CI: 0.37-0.98). However, none of associations remained statistically significant after correction for multiple testing. These data provide some evidence that genetic variability in IL23R may contribute to rectal cancer risk and should be evaluated in additional studies.     

The NADPH-dependent thioredoxin reductase/thioredoxin system in germinating barley seeds: gene expression, protein profiles, and interactions between isoforms of thioredoxin h and thioredoxin reductase

The NADPH-dependent thioredoxin reductase (NTR)/thioredoxin (Trx) system catalyzes disulfide bond reduction in the cytoplasm and mitochondrion. Trx h is suggested to play an important role in seed development, germination, and seedling growth. Plants have multiple isoforms of Trx h and NTR; however, little is known about the roles of the individual isoforms. Trx h isoforms from barley (Hordeum vulgare) seeds (HvTrxh1 and HvTrxh2) were characterized previously. In this study, two NTR isoforms (HvNTR1 and HvNTR2) were identified, enabling comparison of gene expression, protein appearance, and interaction between individual NTR and Trx h isoforms in barley embryo and aleurone layers. Although mRNA encoding both Trx h isoforms is present in embryo and aleurone layers, the corresponding proteins differed in spatiotemporal appearance. HvNTR2, but not HvNTR1, gene expression seems to be regulated by gibberellic acid. Recombinant HvNTR1 and HvNTR2 exhibited virtually the same affinity toward HvTrxh1 and HvTrxh2, whereas HvNTR2 has slightly higher catalytic activity than HvNTR1 with both Trx h isoforms, and HvNTR1 has slightly higher catalytic activity toward HvTrxh1 than HvTrxh2. Notably, both NTRs reduced Trx h at the acidic conditions residing in the starchy endosperm during germination. Interspecies reactions between the barley proteins and Escherichia coli Trx or Arabidopsis thaliana NTR, respectively, occurred with 20- to 90-fold weaker affinity. This first investigation of regulation and interactions between members of the NTR/Trx system in barley seed tissues suggests that different isoforms are differentially regulated but may have overlapping roles, with HvNTR2 and HvTrxh1 being the predominant isoforms in the aleurone layer.     

Differential gene expression between African American and European American colorectal cancer patients

The incidence and mortality of colorectal cancer (CRC) is higher in African Americans (AAs) than other ethnic groups in the U. S., but reasons for the disparities are unknown. We performed gene expression profiling of sporadic CRCs from AAs vs. European Americans (EAs) to assess the contribution to CRC disparities. We evaluated the gene expression of 43 AA and 43 EA CRC tumors matched by stage and 40 matching normal colorectal tissues using the Agilent human whole genome 4x44K cDNA arrays. Gene and pathway analyses were performed using Significance Analysis of Microarrays (SAM), Ten-fold cross validation, and Ingenuity Pathway Analysis (IPA). SAM revealed that 95 genes were differentially expressed between AA and EA patients at a false discovery rate of ≤5%. Using IPA we determined that most prominent disease and pathway associations of differentially expressed genes were related to inflammation and immune response. Ten-fold cross validation demonstrated that following 10 genes can predict ethnicity with an accuracy of 94%: CRYBB2, PSPH, ADAL, VSIG10L, C17orf81, ANKRD36B, ZNF835, ARHGAP6, TRNT1 and WDR8. Expression of these 10 genes was validated by qRT-PCR in an independent test set of 28 patients (10 AA, 18 EA). Our results are the first to implicate differential gene expression in CRC racial disparities and indicate prominent difference in CRC inflammation between AA and EA patients. Differences in susceptibility to inflammation support the existence of distinct tumor microenvironments in these two patient populations.     

Synchronous global assessment of gene and protein expression in colorectal cancer progression

Well-established models of colorectal cancer progression are based on the idea that the disease evolves through a multistep process involving sequential genetic mutations, suggesting that progression through clinically defined stages should correlate with well-defined patterns of gene expression. The majority of studies to date, however, have assessed these processes one gene and one protein at a time. We report the first comprehensive assessment of both gene and protein expression performed in parallel across progressive stages of human colorectal neoplasia. Remarkably, despite the global nature of the gene expression assessment, very few genes could be linked with certainty to specific proteins through currently available annotations. Furthermore, the correlation of expression between identified genes and proteins was poor. Nevertheless, both produced expression signatures that differentiated normal mucosa and nonmalignant adenomas from each other and from the malignant carcinomas and both produced fairly consistent subclasses of the malignancies, suggesting that a molecular staging might be more appropriate provided that these profiles can be tied to clinical outcome. This is potentially important as clinical staging is widely used as a prognostic indicator and used in the decision to pursue adjuvant therapies.     

Differential expression of the L-plastin gene in human colorectal cancer progression and metastasis.

To identify molecular alterations in the progression of colorectal carcinoma, we analyzed gene expression profiles of colon cancer cell lines derived from primary and metastatic tumors from a single patient. Of 2280 cDNAs investigated using our in-house microarray, the expression of 6 genes (tumor-associated antigen L6, L-plastin, the human homologue of yeast ribosomal protein S28, the B-cell translocation gene, mitochondrial aspartate-aminotransferase, and HLA-A) increased, while that of 2 genes (keratin 5 and phosphoglucomutase) decreased in metastatic-tumor-derived cells compared with primary-tumor-derived cells. Of these genes, we assessed the L-plastin gene, an actin-bundling protein, at the protein level using a tissue microarray consisting of 58 clinically stratified colorectal cancer specimens. Consistent with our microarray results, the expression of L-plastin was significantly correlated with the progression of cancer staging. Therefore, our results suggest that the L-plastin gene is a potential metastatic marker. In addition, combining cDNA microarrays and tissue arrays, as shown here, is thought to facilitate the rapid characterization of candidate biomarkers.     

Comparative proteomic analysis of cysteine oxidation in colorectal cancer patients

Oxidative stress promotes damage to cellular proteins, lipids, membranes and DNA, and plays a key role in the development of cancer. Reactive oxygen species disrupt redox homeostasis and promote tumor formation by initiating aberrant activation of signaling pathways that lead to tumorigenesis. We used shotgun proteomics to identify proteins containing oxidation-sensitive cysteines in tissue specimens from colorectal cancer patients. We then compared the patterns of cysteine oxidation in the membrane fractions between the tumor and non-tumor tissues. Using nano-UPLC-MS^E proteomics, we identified 31 proteins containing 37 oxidation-sensitive cysteines. These proteins were observed with IAM-binding cysteines in non-tumoral region more than tumoral region of CRC patients. Then using the Ingenuity pathway program, we evaluated the cellular canonical networks connecting those proteins. Within the networks, proteins with multiple connections were related with organ morphology, cellular metabolism, and various disorders. We have thus identified networks of proteins whose redox status is altered by oxidative stress, perhaps leading to changes in cellular functionality that promotes tumorigenesis.     

Colorectal cancer risk factors in the detection of advanced adenoma and colorectal cancer

Several risk factors for colorectal cancer (CRC) have been identified. If individuals with risk factors are more likely to harbor cancer or it precursors screening programs should be targeted toward this population. We evaluated the predictive value of colorectal cancer risk factors for the detection of advanced colorectal adenoma in a population based CRC colonoscopy screening program. Data were collected in a multicenter trial conducted in the Netherlands, in which 6600 asymptomatic men and women between 50 and 75 years were randomly selected from a population registry. They were invited to undergo a screening colonoscopy. Based on a review of the literature CRC risk factors were selected. Information on risk factors was obtained from screening attendees through a questionnaire. For each CRC risk factor, we estimated its odds ratio (OR) relative to the presence of advanced neoplasia as detected at colonoscopy. Of the 1426 screening participants who underwent a colonoscopy, 1236 (86%) completed the risk questionnaire. 110 participants (8.9%) had advanced neoplasia. The following risk factors were significantly associated with advanced neoplasia detected by colonoscopy: age (OR: 1.06 per year; 95% CI: 1.03–1.10), calcium intake (OR: 0.99 per mg; 95% CI: 0.99–1.00), positive CRC family history (OR: 1.55 per first degree family member; 95%CI: 1.11–2.16) and smoking (OR: 1.75; 95%CI: 1.09–2.82). Elderly screening participants, participants with lower calcium intake, a CRC family history, and smokers are at increased risk of harboring detectable advanced colorectal neoplasia at screening colonoscopy.     

Searching for consistently reported up- and down-regulated biomarkers in colorectal cancer: a systematic review of proteomic studies

The cumulative lifetime risk for the development of colorectal cancer in the general population is 6 %. In many cases, early detection by fecal occult blood test is limited regarding sensitivity. Therefore, there is an urgent need for improved diagnostic tests in colorectal cancer. The recent development of high-throughput molecular analytic techniques should allow the rapid evaluation of new diagnostic markers. However, researchers are faced with an overwhelming number of potential markers form numerous colorectal cancer protein expression profiling studies. To address the challenge, we have carried out a comprehensive systematic review of colorectal cancer biomarkers from 13 published studies that compared the protein expression profiles of colorectal cancer and normal tissues. A protein ranking system that considers the number of comparisons in agreement, total sample sizes, average fold-change and direction of differential expression was devised. We observed that some proteins were consistently reported by multiple studies as differentially expressed with a statistically significant frequency (P < 0.05) in cancer versus normal tissues comparison. Our systematic review method identified proteins that were consistently reported as differentially expressed. A review of the top four candidates revealed proteins described previously as having diagnostic value as well as novel candidate biomarkers. These candidates should help to develop a panel of biomarkers with sufficient sensitivity and specificity for the diagnosis of colorectal cancer in a clinical setting.