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 profiling of cancer of the gingivo-buccal complex: Identification of new differentially expressed markers

Tobacco-related oral cancer is the most common cancer among Indian males, gingivo-buccal complex (GBC) being the most affected subsite due to the habit of chewing tobacco. Proteins from the lysates of microdissected normal and transformed epithelium from clinically well-characterized tissue samples of the GBC were separated by two-dimensional gel electrophoresis to identify differentially expressed proteins. Eleven protein spots showed differential expression, which could withstand the stringency of statistical evaluation. The observations were confirmed with additional tissues. Nine of these differentiators were identified by MS as lactate dehydrogenase B, α-enolase, prohibitin, cathepsin D, apolipoprotein A-I, tumor protein translationally controlled-1, an SFN family protein, 14-3-3σ and tropomyosin. Cluster analysis indicated that these proteins, as a coexpressed set, could distinguish normal and transformed epithelium. Functionally, these differentiator molecules are relevant to the pathways and processes that have been previously implicated in oral carcinogenesis and could therefore be investigated further as a panel of markers for management of cancer of the GBC.     

Large-scale antibody profiling of human blood sera: The future of molecular diagnosis

Despite the progress in cancer diagnosis the timely detection of many cancer types is still a grand challenge. For various human cancer types including lung cancer, prostate cancer, and breast cancer, several groups recently demonstrated that autoantibody profiling might be a promising approach towards earlier and more accurate cancer diagnosis. In this paper, we confirm the ability of autoantibody profiling as a diagnostic test by providing evidence that not only cancer sera can be distinguished well from normal controls, but also from sera of patients with noncancerous diseases. Altogether, we screened blood sera of 191 cancer patients, 60 physiologically unaffected controls, and 177 sera of patients with noncancerous diseases for more than 1800 immunogenic clones. The measured autoantibody fingerprints were evaluated using a novel image analysis pipeline. For 13 antigens, statistically significant (p<0.05) and at least two-fold elevated immuno-reactivity in cancer sera compared to normal sera could be observed. Nine of these antigens also showed increased reactivity compared to sera of patients with other diseases, including the tumor marker vimentin. Supervised discrimination between cancer and normal sera by using linear Support Vector Machines was possible with an accuracy of 94.04%, a specificity of 83.38%, and a sensitivity of 97.44%. Here, our so-called MIMM (minimally invasive multiple marker) approach showed no significant difference in the classification accuracy between low and higher tumor grades. The classification in healthy and diseased sera showed an even higher accuracy of 96.12% while the discrimination in cancer sera and diseased controls revealed an accuracy of 69.58%. These results demonstrate that autoantibody profiling offers the possibility of cancer screening for a variety of different cancer types as well as inflammatory diseases at an early disease stage.      

Identification of nasopharyngeal carcinoma antigens that induce humoral immune response by proteomic analysis

A proteomics-based approach has been used to identify proteins that commonly elicit a humoral immune response in nasopharyngeal carcinoma (NPC). Sera from 19 newly diagnosed NPC patients and 19 healthy individuals were analyzed for IgG autoantibodies against NPC proteins resolved by 2-DE. Protein spots that exhibited selective reactivity with sera from NPC patients were identified by MS. Among nine identified proteins, cytokeratin 19 (CK19), Erb3 binding protein (EBP1), and Rho GDP dissociation inhibitor-beta (Rho-GDI-2) induced autoantibodies in more than 36.8% of NPC patients but not in healthy individuals. Furthermore, Western blot analysis and immunohistochemical staining were performed to determine the expression and localization of CK19, EBP1, and Rho-GDI-2 in NPC and normal nasopharyngeal mucosal tissues. Up-regulated CK19 and EBP1, but not Rho-GDI-2, were observed in NPC vs. normal tissue. Subcellular localization of the three proteins in NPC tissue was same as that in the normal tissue. Thus, overexpression of CK19 and EBP1 may be one of the mechanisms for their autoantibody development in NPC. To validate the findings of a proteomic analysis, occurrence of autoantibodies against these three proteins was detected by immunoprecipitation and Western blot analysis in additional 30 NPC patients, 23 other types of cancer patients and 20 healthy individuals. Results showed that frequency of autoantibodies against CK19, EBP1 and Rho-GDI-2 in NPC patients was significantly higher than that in other types of cancer patients and healthy individuals. We conclude that CK19, EBP1 and Rho-GDI-2 may have utility in NPC screening and diagnosis.     

Studying the protein-protein interactions in the postsynaptic density by means of immunoabsorption and chemical crosslinking

Postsynaptic densities (PSDs), isolated from porcine cerebral cortices, are large disk-shaped aggregates consisting of hundreds of different proteins. To study the protein-protein interactions in such complex supramolecules, we developed a procedure to break up the PSD's overall structure, while preserving some interactions between individual proteins. Using the resulting PSD sample and an indirect immunoabsorption procedure, PSD-95 was isolated along with the α- and β-subunits of calcium calmodulin-dependent protein kinase II (CaMKIIα and CaMKIIβ), α-tubulin, β-tubulin, and Chapsyn110. Similarly, CaMKIIα was isolated along with CaMKIIβ, α-tubulin, β-tubulin, and small amounts of PSD-95. The proteins isolated from PSDs treated with a cleavable bifunctional crosslinking reagent were further subjected to diagonal gel electrophoresis analysis, and the results indicated that CaMKIIα resides next to α-tubulin in the PSD. Overall, the results obtained here suggest that within the PSD, large aggregates of CaMKIIα, CaMKIIβ, α-tubulin, and β-tubulin may occur that indirectly associate with PSD-95 and Chapsyn110. Such a protein organization would allow interactions with F-actin in the cytoplasm and with proteins, such as N-methyl-D-aspartate receptors, which reside on the postsynaptic membrane. Furthermore, it would facilitate binding to proteins such as the various microtubule-associated proteins that reside in the core region of the PSD.     

Structure-guided cancer blockade between bioactive bursehernin and proteins: Molecular docking and molecular dynamics study

Bursehernin (5′-desmethoxyyatein) is a natural lignan, which has anti-tumor activity in vitro. In this study, the binding-inhibitory effects of bursehernin were screening on selected 80 proteins associated with cancer pathway. The computational analysis suggested inhibitory effect due to bursehernin towards proteins related to cancer proliferation, including FMS kinase receptor, heat shock protein 90-α (Hsp90-α), adenylate cyclase 10 (ADCY10), mitogen-activated protein kinase kinase (MEK1), and α-tubulin. Moreover, bursehernin could interfere with cell cycle progression via binding to cyclin B proteins. Among all screened proteins, the compound showed an interesting binding affinity to the FMS kinase receptor. The binding mode studies by molecular dynamic technique showed that aromatic ring of bursehernin compound was responsible for compound-protein interaction through pi-pi stacking with Tyr105 and Phe178 of the FMS kinase receptor. This study suggests that bursehernin has potential for development as an anti-tumor agent with an anti-proliferation, and cell cycle arrest inducing, although further studies are needed.     

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.     

Evaluation of human antibody responses to keyhole limpet hemocyanin on a carbohydrate microarray

Purpose : Keyhole limpet hemocyanin (KLH) is used as a vaccine adjuvant, as a carrier protein for small haptens, and as a treatment for bladder cancer. Immunization with KLH produces antibodies to tumor‐associated carbohydrate antigens (TACAs) in animals, and these antibodies have been postulated as the basis of efficacy for bladder cancer treatment. The purpose of this study was to evaluate antibody responses to KLH in humans. Experimental design : A carbohydrate microarray was used to profile antibody responses in 14 individuals immunized with KLH plus alum adjuvant. Results : Eight out of fourteen individuals produced antibodies to at least one TACA. Increases to Lewis X, Lewis Y, GA1di, GM3, and sialyl Lewis A were observed in certain individuals, but, in general, antibody profiles were highly variable. Pre‐immunization antibody levels to a subset of array antigens had a statistically significant correlation with the magnitude of the antibody response to KLH. Conclusions and clinical relevance : Antibodies to TACAs can be produced in humans, but antibody profiles differ considerably from person to person, which may contribute to variable clinical responses with KLH. Pre‐treatment antibody levels to certain antigens may be useful for predicting which patients will respond favorably to KLH.     

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.     

SEREX, Proteomex, AMIDA, and beyond: Serological screening technologies for target identification

Despite the great body of knowledge about the aetiology, pathogenesis, risk factors, and associated molecular processes, cancer remains a prime health concern. Over the past decades scientific and medical research focused on the identification of biomarkers and target molecules for the diagnosis and therapy of cancer. Such markers may allow for improved and early diagnosis, as well as for immunotherapeutic approaches for cancer treatment. A plethora of technologies dedicated to the identification of target molecules was developed including those relying on a humoral response against tumour-associated antigens (TAA) in diseased individuals. As for other diseases, cancers elicit immune responses that result in the induction of T and B lymphocytes specific for tumour-associated proteins, largely self-antigens, but also those comprising viral and bacterial proteins. Cancer-specific serum antibodies are of great use for the isolation and subsequent identification of their cognate antigens. The present review will concentrate on three major serological target identification methods, i.e. SEREX, Proteomex, and AMIDA, concluding with a summary of the milestones in the clinical advancement and applications of serological TAA.     

Heat-shock protein-27, -70 and peroxiredoxin-II show molecular chaperone function in sickle red cells: Evidence from transgenic sickle cell mouse model

Sickle cell disease (SCD) is an autosomal recessive genetic red cell disorder characterized by the production of a defective form of hemoglobin, hemoglobin-S, that is worldwide-distributed. The acute clinical manifestations of SCD are related to hemoglobin cyclic-polymerization and to the generation of rigid, dense red blood cells (RBCs). We studied RBCs membrane proteome from human sickle RBCs, fractioned according to density compared to normal RBCs. 2-DE followed by MS analysis was carried out. We identified 65 proteins differently expressed, divided into five major clusters according to their functions: (i) membrane-cytoskeleton proteins; (ii) metabolic enzymes; (iii) ubiquitin-proteasome-system; (iv) flotillins; (v) chaperones. HSP27, HSP70 and peroxiredoxin-II (Prx-II) showed the most relevant changes. They were differently recruited to sickle RBCs membrane in response to in vitro hypoxia. Potential markers were then validated in a transgenic-mouse model for SCD, the SAD mice, exposed to hypoxia mimicking acute SCD vaso-occlusive-crisis (VOCs); we found that HSP70 and HSP27 bound to RBCs membrane respectively after 12?h and 48?h of hypoxia, while Prx-II membrane binding was modulated during hypoxia. Our data indicate that HSP27 and HSP70 play a novel role as RBCs membrane protein protectors and as possibly new markers of severity of RBCs membrane damage during acute VOCs.     

An experimental strategy for quantitative analysis of the humoral immune response to prostate cancer antigens using natural protein microarrays

The identification of human tumor antigens has potential utility in the diagnosis and treatment of cancers. We demonstrate here a complete strategy to profile immunoreactivity and identify tumor antigens from proteins derived from tumor cell lines. Microarrays of proteins produced from 2-D LC fractionation of prostate tumor cell-line lysates were used to profile immunoreactivity in the sera of prostate cancer patients and control subjects. Cancer-associated immunoreactivity to distinct groups of chromatography fractions was present in about 50% of the patients, with greater immunoreactivity present in patients with non-organ-confined cancer than in patients with organ-confined cancer. We grouped the immunoreactive fractions by similarities in elution order and patterns of immunoreactivity to guide and interpret the MS analysis of selected fractions, which was used to identify the proteins that may be responsible for the immunoreactivity. As a complementary method to further characterize and validate the immunoreactivity of the proteins identified by mass spectrometry, we demonstrate the use of focused microarrays of recombinant proteins. Disease-associated immunoreactivity was confirmed for one of the identified proteins, human Kallikrein 11. These results demonstrate a practical approach to screening, identifying, and validating immunoreactive proteins that could be applied to diverse studies on humoral immune responses.     

Shotgun immunoproteomics to identify disease‐associated bacterial antigens: Application to human colon cancer

Circulating antibodies reflect a mirror view of invading antigens that are related to infection and cancer. This was recently exemplified by using serum antibodies to capture Streptococcus bovis antigens followed by MS to generate antigen profiles that were diagnostic for colon cancer. These bacterial antigen profiles have a high potential to aid in the immuno‐diagnosis of this disease, as the magnitude of the immune response to bacterial antigens is, in general, superior to the immune response against tumor (self) antigens. In this study, the identity of individual colon cancer‐associated streptococcal antigens was revealed by enrichment of these “diagnostic” antigens by selected patient antibodies followed by high‐accuracy nanoLC‐MS/MS peptide identification. This showed that both the histone‐like protein HlpA and the ribosomal protein Rp L7/L12 are members of the colon cancer‐associated S. bovis immunome. Both antigens also seem to belong to the group of anchorless surface proteins, like 14 additional proteins that were co‐identified in S. bovis cell wall extracts. Among these were the known streptococcal anchorless surface proteins GAPDH and Enolase. Taken together, these data show that shotgun immunoproteomics, combining immunocapture in‐line with LC MS/MS, is a convenient approach for the rapid identification of disease‐associated bacterial antigens.     

Proteomic profiling of heat shock proteins: An emerging molecular approach with direct pathophysiological and clinical implications

The HSP family is one of the most ancient and evolutionarily conserved protective protein families found in nature. Originally discovered as inducible molecules capable of maintaining cellular homeostasis against abrupt temperature changes, HSPs were later determined to represent an adaptive physiological response that copes with a variety of different cellular proteotoxic stresses. These physiological molecular chaperones facilitate the synthesis, folding, assembly, trafficking and secretion of specific proteins in various cellular compartments. Most importantly, these proteins guard the whole cell proteome against misfolding and inappropriate aggregation. A series of diversified proteotoxic stresses, including heat, hypoxia/ischemia, free radicals, acidosis, ATP depletion and toxins are capable of inducing a typical cellular stress response characterised by rapid inhibition of overall protein synthesis, with a concomitant dramatic increase in HSP expression. From a pathophysiological point of view, HSP induction has been observed in a wide spectrum of inflammatory and degenerative diseases (from cancer to prion disease by passing to infective and autoimmune diseases) and, intriguingly, overexpression monitoring seems to have potential implications in terms of diagnosis, prognosis and, above all, therapy. Proteomics studies, identifying a series of modification of HSP expression patterns in different diseases, are confirming these promising clinical applications.     

Chaperoning heat shock proteins: Proteomic analysis and relevance for normal and dystrophin-deficient muscle

Molecular chaperones play a key role in normal muscle function and during physiological adaptations to extensive exercise and numerous forms of cellular stress. The various classes of HSPs and related chaperones are also involved in the molecular pathogenesis of a large number of neuromuscular diseases. Several MS-based proteomic studies have recently shown that the expression levels of molecular chaperones are severely altered in dystrophin-deficient muscles. Dystrophin isoform Dp427 (where Dp427 is dystrophin protein of 427 kDa) is a large membrane cytoskeletal protein and its deficiency is the primary underlying cause of Duchenne muscular dystrophy. Current efforts have focused on the establishment of a comprehensive biomarker signature of dystrophinopathy in order to improve diagnostic methods, establish reliable prognostic factors and identify novel therapeutic targets. Following an introduction into the biology of HSPs and their general role in skeletal muscle, this review outlines the proteomic profiling of molecular chaperones in dystrophinopathy. The focus is especially on the molecular fate of HSPs cardiovascular HSP (HSPB7), αBC (HSPB5), HSP70 (HSPA) and HSP90 (HSPC) in dystrophin-deficient muscles and their involvement in progressive muscular dystrophy. Furthermore, the potential usage of distinct chaperones as disease markers of secondary pathobiochemical changes for the evaluation of novel treatment options is discussed.     

Deep depletion of abundant serum proteins reveals low-abundant proteins as potential biomarkers for human ovarian cancer

Epithelial ovarian cancer (EOC) ranks fifth as a cause of cancer deaths in women. Current diagnostic and monitoring markers have limited reliability for the detection of disease. We have tested the possibility of identifying candidate biomarkers present at low nanogram to picogram levels after removing both the 12 most abundant and 77 moderately abundant proteins from serum samples of EOC patients using antibody affinity columns. We showed that this approach allows the identification of proteins that are expressed at nanogram per liter levels in the serum. Using ICAT/MS/MS analysis, we identified 51 proteins that are differentially expressed by at least twofold. These proteins include leucine-rich α-2-glycoprotein, matrix metalloproteinase-9 (MMP-9), inter-α-trypsin inhibitor heavy chain H1, insulin-like growth factor-binding protein 6, insulin-like growth factor-binding protein 3, isoform 1 of epidermal growth factor receptor, angiopoietin-like protein 3 (ANGPTL3) and phosphatidylcholine-sterol acyltransferase. We confirmed the differential expression of MMP9 and ANGPTL3 in normal and ovarian cancer sera by ELISA assays. Further robust clinical evaluation of the candidate markers identified is necessary.     

Analysis of the human immune response to vaccinia by use of a novel protein microarray suggests that antibodies recognize less than 10% of the total viral proteome

Control of smallpox by mass vaccination was one of the most effective public health measures ever employed for eradicating a devastating infectious disease. However, new methods are needed for monitoring smallpox immunity within current vulnerable populations, and for the development of replacement vaccines for use by immunocompromized or low-responding individuals. As a measure for achieving this goal, we developed a protein microarray of the vaccinia virus proteome by using high-throughput baculovirus expression and purification of individual elements. The array was validated with therapeutic-grade, human hyperimmune sera, and these data were compared to results obtained from individuals vaccinated against smallpox using Dryvax. A high level of reproducibility with a very low background were apparent in repetitive assays that confirmed previously reported antigens and identified new proteins that may be important for neutralizing viral infection. Our results suggest that proteins recognized by antibodies from all vaccinees constituted <10% of the total vaccinia proteome.     

Targeted detection of prostate cancer proteins in serum using heavy‐isotope‐labeled‐peptide standards and MALDI‐TOF/TOF

Proteins released from cancer tissues to patient sera can potentially be used to achieve sensitive, specific, and early detection of cancer by means of blood tests. In this study, we used a platform that combines glycopeptide capture, heavy‐isotope‐labeled‐peptide standards, and liquid chromatography coupled to tandem mass spectrometry to determine which glycoproteins from prostate cancer can be detected in sera from patients with early‐stage prostate cancer. The detection limit for prostate‐specific antigen in serum was 3.44 ng/mL; thus, direct identification of low abundance, cancer‐specific proteins was achieved using our platform. We showed that prostatic acid phosphatase and membrane metallo‐endopeptidase that were detected in sera were preferentially expressed in prostate cancer tissues. Levels of these two proteins were elevated in biopsy‐positive patients but not biopsy‐negative groups. Therefore, these two proteins are candidate biomarkers for analysis of patient samples with levels of prostate‐specific antigen in the diagnostic gray zone.     

Prostate cancer serum biomarker discovery through proteomic analysis of alpha‐2 macroglobulin protein complexes

Alpha‐2 macroglobulin (A2M) functions as a universal protease inhibitor in serum and is capable of binding various cytokines and growth factors. In this study, we investigated if immunoaffinity enrichment and proteomic analysis of A2M protein complexes from human serum could improve detection of biologically relevant and novel candidate protein biomarkers in prostate cancer. Serum samples from six patients with androgen‐independent, metastatic prostate cancer and six control patients without malignancy were analyzed by immunoaffinity enrichment of A2M protein complexes and MS identification of associated proteins. Known A2M substrates were reproducibly identified from patient serum in both cohorts, as well as proteins previously undetected in human serum. One example is heat shock protein 90 alpha (HSP90α), which was identified only in the serum of cancer patients in this study. Using an ELISA, the presence of HSP90α in human serum was validated on expanded test cohorts and found to exist in higher median serum concentrations in prostate cancer (n = 18) relative to control (n = 13) patients (median concentrations 50.7 versus 27.6 ng/mL, respectively, p = 0.001). Our results demonstrate the technical feasibility of this approach and support the analysis of A2M protein complexes for proteomic‐based serum biomarker discovery.