Abstract
African American (AA) men suffer from a disproportionately high incidence and mortality of prostate cancer (PCa) compared with other racial/ethnic groups. Despite these disparities, African American men are underrepresented in clinical trials and in studies on PCa biology and biomarker discovery. We used immunoseroproteomics to profile antitumor autoantibody responses in AA and European American (EA) men with PCa, and explored differences in these responses. This minimally invasive approach detects autoantibodies to tumor-associated antigens that could serve as clinical biomarkers and immunotherapeutic agents. Sera from AA and EA men with PCa were probed by immunoblotting against PC3 cell proteins, with AA sera showing stronger immunoreactivity. Mass spectrometry analysis of immunoreactive protein spots revealed that several AA sera contained autoantibodies to a number of proteins associated with both the glycolysis and plasminogen pathways, particularly to alpha-enolase (ENO1). The proteomic data is deposited in ProteomeXchange with identifier PXD003968. Analysis of sera from 340 racially diverse men by enzyme-linked immunosorbent assays (ELISA) showed higher frequency of anti-ENO1 autoantibodies in PCa sera compared with control sera. We observed differences between AA-PCa and EA-PCa patients in their immunoreactivity against ENO1. Although EA-PCa sera reacted with higher frequency against purified ENO1 in ELISA and recognized by immunoblotting the endogenous cellular ENO1 across a panel of prostate cell lines, AA-PCa sera reacted weakly against this protein by ELISA but recognized it by immunoblotting preferentially in metastatic cell lines. These race-related differences in immunoreactivity to ENO1 could not be accounted by differential autoantibody recognition of phosphoepitopes within this antigen. Proteomic analysis revealed differences in the posttranslational modification profiles of ENO1 variants differentially recognized by AA-PCa and EA-PCa sera. These intriguing results suggest the possibility of race-related differences in the antitumor autoantibody response in PCa, and have implications for defining novel biological determinants of PCa health disparities.
Highlights
From the ‡Center for Health Disparities and Molecular Medicine, Department of Basic Sciences, Loma Linda University School of Medicine, Loma Linda, California 92350; §Department of Biological Sciences, University of Texas, El Paso, Texas 79968; ¶Mass Spectrometry Core Facility, Division of Biochemistry, LLU School of Medicine, Loma Linda, California 92350; ʈLLU Cancer Center Biospecimen Laboratory, Loma Linda, California 92350; **LLU School of Behavioral Health, Loma Linda, California 92350; ‡‡Department of Biology and Center for Cancer Research, Tuskegee University, Tuskegee Alabama 36088; §§Department of Medicine, Division of Rheumatology, LLU School of Medicine, Loma Linda, California 92350
AA men are at a higher risk of developing aggressive Prostate cancer (PCa) compared with other racial/ethnic groups, this population has been underrepresented in most research studies on antitumor autoantibody profiling in PCa
Given the heterogeneity of PCa tumors in diverse male populations, and the recent reports pointing to racial differences in the immunobiology of prostate tumors [32, 36, 38], we undertook an effort to profile, using an immunoseroproteomics approach, sera from PCa patients of African and European descent for the presence of autoantibodies to potential tumor-associated antigens (TAAs)
Summary
Study Subjects and Sample Collection—Sera from patients with PCa (n ϭ 59, self-identified AA, median age 71 yr (average 70); n ϭ 98, self-identified EA, median age 66 yr (average 67)), were obtained from Loma Linda University (LLU) Cancer Center Biospecimen Laboratory, Bioserve (Beltsville, MD), LLU Medical Center, and the serum bank in the LLU Center for Health Disparities and Molecular Medicine. Analysis 2D gels loaded with 0.5 mg of total protein from cell lysates were processed for protein transfer to PVDF membranes and subsequent Western blotting (WB) with human sera as described below. Sample Preparation for Mass Spectrometry—Digital images from the reference 2D gel (Coomassie Blue-stained) and analysis 2D gel (immunoblot film) were acquired using a Licor-Odyssey scanner and overlaid with Adobe Photoshop 7.0 software using the reference circle marks drawn around prominent protein spots in the PVDF membranes for accurate alignment. The final filtering process of the top scores of candidate TAA hits included identification from multiple sera, the correct corresponding molecular weight and PI in the gel/film, percent coverage, number of unique peptides, and the total number of proteins identified (supplemental Table S1). A p value of 0.05 or less was accepted as a determinant for statistical significance
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