Identification of tumor antigens in renal cell carcinoma by serological proteome analysis

Abstract

We have investigated the suitability of proteomics for identification of tumor-associated antigens. First, we compared the proteomes of nontumorous kidney and renal cell carcinoma (RCC) by two-dimensional gel electrophoresis (2-DE) and silver staining. Protein patterns were markedly different (approximately 800 spots in RCCs versus approximately 1400 spots in kidney). 2-DE immunoblotting revealed five RCC-specific spots, reproducibly reactive with RCC-patient but not healthy donor control sera. Two of these antigens were isolated by preparative 2-DE, and identified by Edman sequencing of tryptic peptides. The first antigen, smooth muscle protein 22-alpha (SM22-alpha), is an actin-binding protein of unknown function predominantly expressed in smooth muscle cells. In situ hybridization revealed that SM22-alpha is not expressed in the malignant cells but in mesenchymal cells of the tumor stroma. The second antigen represents carbonic anhydrase I (CAI), an isoform usually not expressed in kidney. Interestingly, a different isoform (CAXII) has previously been identified by serological expression cloning as an antigen overexpressed in some RCCs. In additional assays, antibodies to recombinant CAI or SM22-alpha were detected in sera from 3/11 or 5/11 RCC patients, respectively, whereas sera from 13 healthy individuals did not react. In conclusion, serological proteome analysis may be a new tool for the identification of tumor-associated antigens.

Tumor-associated Antigen Arrays for the Serological Diagnosis of Cancer

“…The combination of standard proteomic tools of 2D gels and MS with serological screening of 2D gel-derived Western blots is an emerging approach that promises to yield a high number of candidate TAAs (8,134,135). This approach has been termed "serological proteome analysis" (SERPA) (134).…”

“…This approach has been termed "serological proteome analysis" (SERPA) (134). In this approach, proteins in lysates prepared from tumors or cancer cell lines are separated by 2D gel electrophoresis and transferred to membranes that are then incubated with individual or pooled sera from cancer patients or healthy controls.…”

“…Immunoreactive proteins that exhibit specific reactivity with cancer sera are then identified by MS. Various groups have used SERPA to identify candidate TAAs associated with breast cancer, including the RNA-binding protein regulatory subunit (RS), DJ-1 oncogene, glucose-6-phosphate dehydrogenase, heat shock 70-kDa protein 1 (HS71), and dihydrolipoamide dehydrogenase (8,29,134,135). Interestingly, although glucose-6-phosphate dehydrogenase is widely expressed in normal cells, the breast cancer sera reacted preferentially with some isoforms of the protein detected by MS, suggesting that autoantibodies in these sera might be driven by posttranslational modifications (8), which is consistent with previous observations that intracellular proteins targeted by autoantibodies in systemic autoimmune diseases often undergo these modifications (41).…”

Cancer Immunomics Using Autoantibody Signatures for Biomarker Discovery

“…More recently, proteomics, using a combination of sophisticated methods, provided new opportunities for screening and identifying autoantigens (12,18,(21)(22)(23). Based on the different combinations that can be useful for immunomics, different types of workflows corresponding to those currently used in proteomics can be proposed (Fig.…”

Étude de la réaction immunitaire humorale aux cancers par des approches protéomiques

“…Dans cette dernière étude, les annexines I et II ont été identifiées comme des antigènes tumoraux induisant une réponse humorale chez plus de 60 % des patients atteints d'adénocarcinome. Cette approche, qui a été nommée par la suite SERPA (serological proteome analysis) [18], Proteomex [19,20] ou SPEAR (serological and proteomic evaluation of antibody response) [21], a été utilisée avec succès pour identifier des AAT dans plusieurs autres types de cancers. Sans vouloir être exhaustif, on peut citer, par exemple, la protéine peroxirédoxine 6 dans le carcinome squameux de l'oesophage [22,23], les protéines triosephosphate isomérase (Tim) et superoxyde dismutase (MnSOD) dans le carcinome squameux du poumon [22,23], la calréticuline dans les cancers hépatique [24] et du pancréas [25] et RS/DJ1 dans le cancer du sein [26].…”