Proteomics-based identification of HSP60 as a tumor-associated antigen in colorectal cancer

Patients with cancer frequently develop autoantibodies. The identification of tumor autoantigens may have utility in early cancer diagnosis and immunotherapy. In this study, we used serological proteomics analysis (SERPA) to identify tumor proteins that elicit humoral response in colorectal cancer (CRC). The CRC cell line HCT116 was used as a source of proteins for 2-DE and subsequent Western blot analysis in which individual serum from patients with CRC was analyzed for autoantibodies. An autoantibody against HSP60 identified by MS was detected in 13 out of 25 patients with CRC and 1 out of 15 healthy subjects. In addition, the HSP60 expressions in tumor tissues collected from 40 patients with CRC were assessed by immunohistochemistry, and serum specimens from 100 patients with cancer and 30 healthy controls were screened for antibody titer to HSP60 by ELISA. The results showed that expressions of HSP60 in tumor tissue and serum antibody titer to HSP60 were significantly higher in patients with CRC than in healthy subjects. Thus, we conclude that the SERPA is an excellent assay for the identification of tumor-associated antigens and tumor markers. The detection of HSP60 may have clinical utility in CRC screening, diagnosis, and immunotherapy.     

Proteomic analysis of autoantigens associated with systemic lupus erythematosus: Anti-aldolase A antibody as a potential marker of lupus nephritis

To screen for autoantibodies associated with systemic lupus erythematosus (SLE), we used proteomic approaches combining 2-D PAGE and Western blot analysis, followed by protein identification by LC-MS/MS analysis, resulting in the identification of aldolase A as a novel autoantigen in SLE. ELISA showed the prevalence of anti-aldolase A antibodies to be 29.3% in SLE, 8.2% in rheumatoid arthritis, 18.1% in polymyositis and absent in healthy controls. Furthermore, 43.4% of SLE patients suffering from nephritis showed anti-aldolase A autoantibodies, which was significantly higher than the prevalence for those without nephritis (11.1%). In lupus nephritis, there are few reliable diagnostic methods, other than urinalysis. Therefore, these results indicate that autoantibodies against aldolase A may serve as an alternative clinical biomarker of SLE associated with nephritis.     

2-D DIGE and MALDI-TOF-MS analysis of the serum proteome in human osteosarcoma

We performed 2-D DIGE on proteins prepared from serum obtained from patients with osteosarcoma (OS) and controls, to identify differentially expressed proteins that might serve as serum biomarkers for OS prognosis. Proteins found to be differentially expressed were identified by MALDI-TOF mass spectrometric analysis, coupled with database interrogation. We compared serum samples from four individuals with OS to four age- and sex-matched healthy controls. We identified 24 protein spot-features that were significantly increased, and 34 that were significantly decreased in serum from patients with OS relative to the controls. The MS analysis revealed 18 unique proteins that were increased, and 25 unique proteins that were decreased in OS serum samples. Western blot and ELISA analysis confirmed increased levels of amyloid-related serum protein (SAA) in the OS serum samples. The increased expression levels of SAA were decreased after using MTX and cisplatin combination chemotherapy, and were further decreased after operation. Moreover, increased expression levels of sera SAA were seen in the relapsed patients. Our results suggested that the determination of serum SAA in OS patients might be utilized as a marker for relapse and in evaluation of the efficacy of therapy.     

Proteomic analysis of alpha-1-antitrypsin in immunoglobulin A nephropathy

Immunoglobulin A nephropathy (IgAN) is recognized as the most common form of primary glomerulonephritis worldwide. It is characterized by mesangial cell proliferation with mesangial IgA deposition in the glomeruli, and is usually associated with secondary tubulointerstitial injury. Although significant progress has been made in the clarification of the pathogenesis of IgAN, the exact pathogenetic mechanism remains unclear. To find out the candidate proteins that play an important role in IgAN, renal cortex tissues and urine from IgAN patients were studied. The 2‐DE was performed on renal tissues of IgAN and normal controls. A series of spots identified as alpha‐1‐antitrypsin (AAT) by mass spectrometry, were found to be significantly increased in patients with IgAN. Up‐regulation of AAT variants was validated in renal cortex tissues of IgAN using Western blot and 2‐DE immunoblot. Lower isoforms (?48 kDa) and fragments (?33 kDa), suspected as cleavage forms by proteinase attack, were especially increased in IgAN compared to normal controls. In addition, AAT proteins modified by tyrosine nitration (approximately 57 and 48 kDa), which reflects excessive oxidative stress, were increased in IgAN tissue. Additionally in the urine of IgAN, increase of AAT variants and fragments was detected by 2‐DE immunoblot as well as Western blot. Immunohistochemical staining of IgAN kidney tissue revealed that the increase of AAT appeared to be derived from hypertrophic proximal tubules. The AAT staining in the glomerulus was not clear in IgAN. In addition, immunodepletion‐zymography showed a positive correlation between AAT and 80–110‐kDa proteinases in IgAN tissue. Further studies regarding the functional roles of AAT and the proteinases will allow better understanding of the pathogenesis of IgAN.     

Identification of anti-α-enolase autoantibody as a novel serum marker for endometriosis

Diagnosis of endometriosis needs invasive maneuvers. New serum marker that possesses both high sensitivity and high specificity has long been desired. To establish novel serum marker for endometriosis, serum autoantibodies (autoAbs) were investigated using proteomic approach. AutoAbs in sera of endometriotic patients and healthy controls were analyzed using a mesothelial cell line, 2-DE and Western blotting. Proteins in reacted spots were identified using MALDI TOF-MS with MASCOT analysis. ELISAs were established using recombinant proteins and autoAb-titers were estimated in sera of endometriotic patients, disease and healthy controls. Several autoAbs were identified. Anti-α-enolase (Eno1)-autoAb levels in endometriotic patients were significantly elevated compared with both healthy and disease controls. Sensitivity and specificity of serum anti-Eno1-autoAb was nearly comparable to serum CA125. When anti-Eno1-autoAb and CA125 assays were combined, diagnostic sensitivity and accuracy improved. Serum anti-Eno1-autoAb can be a new serum endometriotic marker and it is useful as a supplement assay for CA125. This study validates further clinical evaluation of this novel marker.     

Towards a comprehensive proteome of normal and malignant human colon tissue by 2-D-LC-ESI-MS and 2-DE proteomics and identification of S100A12 as potential cancer biomarker

The aim of this study was to characterize the proteome of normal and malignant colonic tissue. We previously studied the colon proteome using 2‐DE and MALDI‐MS and identified 734 proteins (Roeßler, M., Rollinger, W., Palme S., Hagmann, M.‐L., et al.., Clin. Cancer Res. 2005, 11, 6550–6557). Here we report the identification of additional colon proteins from the same set of tissue samples using a complementary nano‐flow 2‐D‐LC‐ESI‐MS. In total, 484 proteins were identified in colon. Of these, 252 had also been identified by the 2‐DE/MALDI‐MS approach, whereas 232 proteins were unique to the 2‐D‐LC‐ESI‐MS analysis. Comparing protein expression in neoplastic and normal colon tissue indicated elevated expression of several proteins in colorectal cancer, among them the well established tumor marker carcinoembryonic antigen, as well as calnexin, 40S ribosomal protein S15a, serpin H1, and S100A12. Overexpression of these proteins was confirmed by immunoblotting. Serum levels of S100A12 were determined by ELISA and were found to be strongly elevated in colorectal cancer patients compared to healthy individuals. We conclude, that 2‐D‐LC‐ESI‐MS is a powerful approach to identify and compare protein profiles of tissue samples, that it is complementary to 2‐DE/MALDI‐MS approaches and has the potential to identify novel biomarkers.     

An application of the 2-nitrobenzenesulfenyl method to proteomic profiling of human colorectal carcinoma: A novel approach for biomarker discovery

In the development of novel biomarkers, the proteomic approach is advantageous because using it the cancer-associated proteins can be directly identified. We previously developed a 2-nitrobenzenesulfenyl (NBS) method to improve quantitative proteome analysis. Here, we applied this method to proteomic profiling of colorectal carcinoma (CRC) to identify novel proteins with altered expression in CRC. Each pair of tumor and normal tissue specimens from 12 CRC patients was analyzed, and approximately 5000 NBS-labeled paired peaks were quantified. Peaks with altered signal intensities (>1.5-fold) and occurring frequently in the samples (>70%) were selected, and 128 proteins were identified by MS/MS analyses as differentially expressed proteins in CRC tissues. Many proteins were newly revealed to be CRC related; 30 were reported in earlier studies of CRC. Six proteins that were up-regulated in CRC (ZYX, RAN, RCN1, AHCY, LGALS1, and VIM) were further characterized and validated by Western blot and immunohistochemistry. All six were found to be CRC-localized, either in cancer cells or in stroma cells near the cancer cells. These results indicate that the proteins identified in this study are novel candidates for CRC markers, and that the NBS method is useful in proteome mining to discover novel biomarkers.     

A unified sample preparation protocol for proteomic and genomic profiling of cervical swabs to identify biomarkers for cervical cancer screening

Cervical cancer screening is ideally suited for the development of biomarkers due to the ease of tissue acquisition and the well-established histological transitions. Furthermore, cell and biologic fluid obtained from cervix samples undergo specific molecular changes that can be profiled. However, the ideal manner and techniques for preparing cervical samples remains to be determined. To address this critical issue a patient screening protein and nucleic acid collection protocol was established. RNAlater was used to collect the samples followed by proteomic methods to identify proteins that were differentially expressed in normal cervical epithelial versus cervical cancer cells. Three hundred ninety spots were identified via 2-D DIGE that were expressed at either higher or lower levels (>three-fold) in cervical cancer samples. These proteomic results were compared to genes in a cDNA microarray analysis of microdissected neoplastic cervical specimens to identify overlapping patterns of expression. The most frequent pathways represented by the combined dataset were: cell cycle: G2/M DNA damage checkpoint regulation; aryl hydrocarbon receptor signaling; p53 signaling; cell cycle: G1/S checkpoint regulation; and the ER stress pathway. HNRPA2B1 was identified as a biomarker candidate with increased expression in cancer compared to normal cervix and validated by Western blot.     

Automatic Tumor Segmentation with Deep Convolutional Neural Networks for Radiotherapy Applications

Accurate tumor delineation in medical images is of great importance in guiding radiotherapy. In nasopharyngeal carcinoma (NPC), due to its high variability, low contrast and discontinuous boundaries in magnetic resonance images (MRI), the margin of the tumor is especially difficult to be identified, making the radiotherapy planning a more challenging problem. The objective of this paper is to develop an automatic segmentation method of NPC in MRI for radiosurgery applications. To this end, we present to segment NPC using a deep convolutional neural network. Specifically, to obtain spatial consistency as well as accurate feature details for segmentation, multiple convolution kernel sizes are employed. The network contains a large number of trainable parameters which capture the relationship between the MRI intensity images and the corresponding label maps. When trained on subjects with pre-labeled MRI, the network can estimate the label class of each voxel for the testing subject which is only given the intensity image. To demonstrate the segmentation performance, we carry on our method on the T1-weighted images of 15 NPC patients, and compare the segmentation results against the radiologist’s reference outline. Experimental results show that the proposed method outperforms the traditional hand-crafted features based segmentation methods. The presented method in this paper could be useful for NPC diagnosis and helpful for guiding radiotherapy.     

Increased expression of annexin I is associated with drug-resistance in nasopharyngeal carcinoma and other solid tumors

Adjuvant chemotherapy alongside radiotherapy is one of the effective therapies in nasopharyngeal carcinoma (NPC) treatment. However, the appearance of drug resistance is a major obstacle for anti-cancer chemotherapy and often causes failure of the chemotherapy. In this study, a drug-resistant gene annexin I (ANX-I) was identified by comparing differentially expressed proteins between a cisplatin (CDDP)-resistant NPC cell line CNE2-CDDP and parental CNE2 cells using 2-DE. When ANX-I was transfected into CNE2 cells, the CDDP resistance of CNE2 cells was dramatically increased. The drug-resistant ability of ANX-I was demonstrated by both in vitro and in vivo assays. The association of ANX-I expression with clinical features was also investigated. Increased expression of ANX-I was significantly associated with disease relapse in NPC (p<0.05). In breast and gastric cancer, increased expression of ANX-I was significantly associated with drug resistance (p<0.001) and poor prognosis (p<0.001), respectively. Taken together, our findings suggest that ANX-I plays an important role in drug resistance.     

Differences in the proteome profile in placenta from normal term and preeclamptic preterm pregnancies

The aim of this study was to use proteomic approaches to examine differences in protein expression in placentae from normal term and preterm preeclamptic pregnancies and to validate the data thus obtained by other independent methods. Using 2-DE we found that 80% of the proteins were present in both normal and preeclamptic placentae. However, 26 proteins in the normal term placentae were not matched in the preterm preeclamptic group. Six proteins showed increased intensity and one protein was down-regulated in preeclampsia. Four of the seven proteins that were altered in preeclampsia were further analyzed by Western blot and immunohistochemistry. Identification by MS techniques revealed these proteins to be involved in regulatory pathways activated by stress. This is significant because preeclampsia is a multisystem disorder in human pregnancies that results in considerable oxidative and nitrative stress. Three proteins identified by MS to be Hsp27, catalase, and glucose-regulated protein were confirmed by Western blot analysis to be significantly up-regulated in preeclampsia. Endothelial monocyte-activating polypeptide was shown to be down-regulated in preeclampsia by 2-DE and MS.     

Proteomics analysis of plasma for potential biomarkers in the diagnosis of Alzheimer's disease

The objective of this study was to search for biological markers associated with Alzheimer's disease (AD). Plasma specimens obtained from ten pathologically diagnosed AD patients and ten non-demented (ND) control subjects were analyzed by a combination of 2-DE and MS. This strategy allowed us to identify six plasma proteins (alpha-1-antitrypsin, vitamin D-binding protein, inter-alpha-trypsin inhibitor family heavy chain-related protein, apolipoprotein J precursor, cAMP-dependent protein kinase catalytic subunit alpha 1, and an orf) whose 2-DE spot densities were different between the AD and ND groups. Due to their involvements in AD amyloid plaque formation, the plasma concentrations of alpha-1-antitrypsin and apolipoprotein J were further validated using either ELISA or Western blot. The results revealed that the plasma levels of alpha-1-antitrypsin in AD were higher than those of controls, confirming the 2-DE findings. However, no difference in total apolipoprotein J concentration was observed between the AD and ND groups. Considering the difference in resolving power to differentially quantitate protein isoforms provided by 2-DE and Western blot, 2-DE analysis combined with MS protein identification offers distinctive advantages when a disease-related protein isoform-specific variance is investigated.     

Analysis of the differential proteome of human erythroblasts during in vitro erythropoiesis by 2-D DIGE

In the present study we have used an in vitro culture system that induces differentiation of human CD34(+) cells down the erythroid lineage along with 2-D DIGE to determine the differential proteome of erythroblasts at specific developmental stages during erythropoiesis. We initially distinguished 154 proteins differentially expressed between pro-normoblasts and polychromatic/orthochromatic erythroblasts, of which 24 protein spots, representing 21 different proteins, were identified following MS/MS and verification in replicate experiments with cells from different individuals. These data were confirmed by analysis of the differential proteome of erythroblasts at more discrete stages of erythropoiesis using 2-D DIGE and by mapping the expression of three identified proteins (Annexin I, Annexin II, Carbonic Anhydrase I) throughout erythropoiesis by Western blot with specific antisera. In addition, the differential expression of proteins due to biological variation, such as polymorphism, was determined by comparing erythroblasts at the same developmental stage from different individuals; none of the proteins thus identified were represented in the above data set. Finally, we discuss the problems associated with 2-D DIGE gel-based proteomic approaches such as ours and suggest a modified approach for decreased inter-gel variation, improved protein resolution and increased protein concentration, which should significantly facilitate protein identification.     

Comparative proteomic analysis to discover potential therapeutic targets in human multiple myeloma

To clarify the molecular mechanisms that participate in the formation of multiple myeloma (MM) and to detect any tumor-related biomarkers, we performed proteomic analysis of cellular protein extracts from MM cells and normal plasma cells. Plasma cells from nine patients with newly diagnosed MM and nine healthy donors were purified by using anti-CD138 based immunomagnetic bead-positive selection. The protein profiles of purified MM and normal plasma cells were compared using 2-DE. We identified a total of 43 differentially expressed proteins, and confirmed with Western blotting six proteins. The altered proteins were analyzed using the software program Pathway Studio and the biological network can be accessed via (http://life-health.jnu.edu.cn/pathway/pathway.html). Further functional studies showed that annexin A1 knock down modestly induces lethality alone and potentiates the effects of dexamethasone on both dexamethasone-sensitive and dexamethasone-resistant MM cells. By correlating the proteomic data with these functional studies, the current results provide not only new insights into the pathogenesis of MM but also direct implications for the development of novel anti-MM therapeutic strategies and could lead to the discovery of potential therapeutic targets. Future molecular and functional studies would provide novel insights into the roles of these dysregulated proteins in the molecular etiology of MM.     

Differential expression of AQP1 in microdomain-enriched membranes of renal cell carcinoma

Human aquaporin-1 (AQP1) is the most studied member of the aquaporin family, acting as molecular water channel. It is also considered a differentiation marker for proximal renal tubular cells, from which clear cells renal cell carcinoma (RCC) originates, playing an important role in urine formation. We therefore studied AQP1 expression at the proteomic level in RCC and normal tissues, mainly focusing on microdomain-enriched membranes in which AQP1 is highly concentrated. Subcellular fractions were prepared through differential centrifugation, and microdomain-enriched fractions were purified from a plasma membrane-enriched fraction by 1% Triton X-100 treatment followed by ultracentrifugation in sucrose gradient. After SDS-PAGE and Western blot analyses with antibodies against AQP1, lower expression levels of AQP1 isoforms were observed in each subcellular fraction of RCC compared to fractions from normal kidney tissues. The presence of AQP1 in the immunoreactive bands was verified by MALDI-TOF-MS and LC-ESI-MS/MS analysis. Glycosylation of AQP1 was also investigated using N-glycosidase F, confirming the presence of a N-glycosylated isoform of AQP1 in the 35-45-kDa region. These results highlight an under-expression of AQP1 protein and its glycosylated isoforms in homogenate and subcellular fraction obtained from RCC tissue compared to adjacent normal cortex.     

Decreased annexin A3 expression correlates with tumor progression in papillary thyroid cancer

Purpose : The aim of this study is to identify the potential tumor markers that function in carcinogenesis and tumor progression, thus providing important diagnostic and prognostic information. Experimental design : We performed 2‐D gel electrophoresis and MALDI‐TOF MS to investigate the differentially expressed proteins in 25 papillary thyroid carcinoma tissues. For validation of candidate proteins and investigation of clinical significance, we performed Western, Northern blot analysis and immunohistochemical staining. Results : Our proteomic analyses revealed significantly decreased annexin A3 expression in papillary thyroid carcinoma at both the protein and mRNA levels, compared with normal thyroid tissue. ANXA3 immunoreactivity was not significantly correlated with lymph node metastasis, multifocality, capsular invasion or perithyroidal extension in thyroid cancer. However, the tumor subgroup with a lymph node metastasis score of >3 displayed significantly lower ANXA3 expression than did subgroups with negative and ≤3 scores (p=0.001). Moreover, ANXA3 expression was markedly lower in large tumors (>1 cm in diameter) than in microcarcinomas (p=0.001). Conclusion and clinical relevance : Decreased expression of ANXA3 in papillary thyroid cancer supports the idea that ANXA3 may be an effective marker of microcarcinoma, and a negative predictor of papillary thyroid cancer progression.     

Identification of circulating endorepellin LG3 fragment: Potential use as a serological biomarker for breast cancer

Comparative proteome analysis was performed on the cultured media of human nontumor and malignant breast cell lines, Hs578Bst and Hs578T, respectively, in search of a serological biomarker(s) for breast cancer. Proteins in the conditioned media were separated by 2‐D PAGE and then visualized by silver‐staining. Eight proteins changed differentially by more than two‐fold were identified by MALDI‐TOF/TOF MS. Among the proteins identified, the terminal laminin‐like globular (LG3) domain of endorepellin, which was recently reported as an antiangiogenesis factor, was decreased in the cancer cell line. We confirmed the bone morphogenic protein‐1 (BMP‐1) mediated cleavage site on the N‐terminus of endorepellin LG3 fragment. This finding suggests that the LG3 fragment is specifically released by a BMP‐1 driven limited proteolytic process. The protein was also detected in plasma by Western blot analysis and selected reaction monitoring (SRM). The plasma level of the endorepellin LG3 fragment was significantly lower in breast cancer patients compared to healthy donors (p = 0.017; n = 12). The LG3 protein concentration in the control plasma was measured at approximately 3.7 pmol/mL compared to 1.8 pmol/mL in plasma from the cancer patients. We suggest that these results support the potential use of the endorepellin LG3 fragment as a new serological biomarker for breast cancer.     

Intrinsic subtype-associated changes in the plasma proteome in breast cancer

Breast cancers are classified into five intrinsic subtypes: Luminal subtype A, Luminal subtype B, HER2+, Basal, and Normal-like. In this study, we compared the plasma proteome of patients with Luminal A, Luminal B, HER2+, and Basal subtype with plasma from healthy individuals. Protein changes were considered significant if q-value (false discovery rate) was less than 5%. The highest number of changes in the plasma proteome was observed in patients with Luminal type B followed by Basal type breast cancers. The plasma proteome of Luminal A and HER2+ breast cancer patients did not differ significantly from healthy individuals. In Basal breast cancer, a significant number of plasma proteins were downregulated compared with healthy individuals. Acute phase-response proteins α-glycoprotein orosomucoid 1 and serum amyloid protein P were specifically upregulated in the plasma of Luminal B breast cancer patients, suggesting prevalence of low-grade inflammation. Proteins involved in immune response and free radical scavenging were downregulated in the plasma of Luminal B patients, which is in agreement with defective immune system observed in cancer patients. These results reveal intrinsic subtype specific changes in the plasma proteome that may influence tumor progression as well as the systemic effects of cancer.     

Detection of potential markers of primary fibromyalgia syndrome in human saliva

In the last few years, many attempts have been carried out for the research of specific biological biomarkers in fibromyalgia (FM) since, so far, no laboratory tests have been appropriately validated for the diagnosis and the prognostic stratification of the disease. In our study for the first time, we carried out a proteomic analysis of the whole saliva of FM patients in order to evaluate salivary biomarkers. Twenty-two FM patients with all fulfilling the American College of Rheumathology diagnostic criteria for FM and 26 sex-and age-matched healthy subjects were enrolled in the study. Proteomic analysis was performed by combining 2-DE and MALDI-TOF-MS. The most relevant observation which emerged from the data analysis was the exclusive and significant over-expression of transaldolase and phosphoglycerate mutase I. These findings were validated by Western blot analysis and the total optical density confirmed the significant up-regulation of transaldolase and phosphoglycerate mutase I in FM samples with respect to healthy subjects. It was noteworthy that seven further salivary proteins resulted differentially expressed, namely: calgranulin A, calgranulin C, cyclophilin A, profilin 1, Rho GDP-dissociation inhibitor 2, proteasome subunit-α-type-2 and haptoglobin-related protein precursor. These preliminary results demonstrated the utility of salivary proteomic analysis in the identification of salivary biomarkers in FM patients and in clarifying some of the pathogenetic aspects of the disease.     

Toward a better understanding of preeclampsia: Comparative proteomic analysis of preeclamptic placentas

Preeclampsia (PE), a pregnancy-specific syndrome of hypertension, proteinuria, and other systemic disturbances, is a state of widespread endothelial dysfunction secondary to defective placentation. Morphologically, the current data displayed degenerative and apoptotic changes in the mitochondria and villous trophoblasts of preeclamptic placenta. To reveal the superimposing alterations in placental proteins that might explain the pathophysiology of PE, we performed 2-DE MALDI-TOF MS/MS proteomics analysis of differentially expressed placental proteins with placenta from eight normal and eight preeclamptic pregnancies. The identified proteins were confirmed by Western blot analysis. We also performed morphologic evaluation of preeclamptic placentas under both electron and light microscopy. The results disclosed the marked overexpression of chaperonin 60, GST, VDAC, ERp29, and cathepsin D in PE. These proteomics findings clearly suggest the possible cellular battle against mitochondria-originated oxidative stress during PE that either end up with recovery or apoptosis. These results provide a better understanding of proteomic alterations and may help in clarification of stress-related changes in preeclamptic placentas.