Identification of a Novel Autoimmune Peptide Epitope of Prostein in

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Identification Of A Novel Autoimmune Peptide Epitope Of Prostein In Prostate Cancer Elisa Pin, Frauke Henjes, Mun-Gwan Hong, Fredrik Wiklund, Patrik Magnusson, Anders Bjartell, Mathias Uhlén, Peter Nilsson, and Jochen M Schwenk J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00620 • Publication Date (Web): 04 Oct 2016 Downloaded from http://pubs.acs.org on October 6, 2016

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Journal of Proteome Research

Identification Of A Novel Autoimmune Peptide Epitope Of Prostein In Prostate Cancer AUTHOR NAMES Elisa Pin#, Frauke Henjes#, Mun-Gwan Hong#, Fredrik Wiklund‡, Patrik Magnusson‡, Anders Bjartell§ , Mathias Uhlén#, Peter Nilsson#, and , Jochen M. Schwenk*#

AUTHOR ADDRESS #Affinity Proteomics, SciLifeLab, School of Biotechnology, KTH - Royal Institute of Technology, Stockholm, Sweden. ‡Department of Medical Epidemiology and Biostatistics (MEB), Karolinska Institutet; Stockholm, Sweden. § Department of Translational Medicine, Division of Urological Cancers, Skåne University Hospital Malmö, Lund University, Sweden

KEYWORDS Autoimmunity, prostate cancer, prostein, planar microarray, suspension bead array, profiling, epitope mapping, Human Protein Atlas, antigen, peptide

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ABSTRACT There is a demand for novel targets and approaches to diagnose and treat prostate cancer (PCA). In this context, serum and plasma samples from a total of 609 individuals from two independent patient cohorts were screened for IgG reactivity against a sum of 3,833 human protein fragments. Starting from planar protein arrays with 3,786 protein fragments to screen 80 patients with and without PCA diagnosis, 161 fragments (4%) were chosen for further analysis based on their reactivity profiles. Adding 71 antigens from literature, the selection of antigens were corroborated for their reactivity in a set of 550 samples using suspension bead arrays. The antigens prostein (SLC45A3), TATA-box binding protein (TBP) and insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) showed higher reactivity in PCA patients with late disease compared to early disease. Due to its prostate tissue specificity, we focused on prostein and continued with mapping epitopes of the 66-mer protein fragment using patient samples. Using bead-based assays and 15-mer peptides, a minimal peptide epitope was identified and refined by alanine-scanning to the KPxAPFP. Further sequence alignment of this motif revealed homology to transmembrane protein 79 (TMEM79) and TGF-beta-induced factor 2 (TGIF2), thus providing a reasoning for crossreactivity found in females. A comprehensive workflow to discover and validated IgG reactivity against prostein and homologous targets in human serum and plasma was applied. This study provides useful information when searching for novel biomarkers or drug targets that are guided by the reactivity of the immune system against autoantigens.


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INTRODUCTION According to WHO, prostate cancer (PCA) is the second most incident cancer in men and the fifth leading cause of death by cancer in men worldwide1. Five-years survival is reached by almost 100% of PCA patients if the disease is still at a local and regional stage, however the survival rate drops down to 30% in case of aggressive form with distant metastasis and relapse after treatment 1. The absence of alternative interventions for aggressive disease often determines patient overtreatment causing important side effects such as incontinence and impotence2. Thus, the identification of new biomarkers for patient stratification would be a major advance in PCA management. To date, and despite the high number of studies, only very few biomarkers for prediction of PCA progression have been verified in different cohorts or using different techniques. Yet none was translated into clinical practice3. PSA still remains the only molecular marker for PCA even though non specific3. Proteomics keeps adding tiles to the molecular picture of PCA development and progression by means of high-throughput and sensitive technologies allowing to detect and quantify proteins in tissue and body fluids4. Serological biomarkers are desirable due to low invasiveness, low cost and standardized handling procedures. Among serological biomarkers are autoantibodies 5. The relatively high frequency of antibody responses against tumorassociated antigens (TAAs) led to the assumption that they could be of use in the clinical setting. Accordingly, a lot of effort was invested in correlating autoantibodies with clinical parameters, aiming to demonstrate their usefulness in diagnosis and prognosis processes 4, 6,7. Prostate cancer is regarded as an immunogenic tumor. In 2000, Wang and colleagues reported an autoimmune signature able to distinguish PCA from controls with high specificity and sensitivity8. More recently, cyclin B1 was reported as a potential marker for 3 ACS Paragon Plus Environment


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PCA immunodiagnosis9. Another study found and validated 174 antigens corresponding to proteins related to the cytoskeleton, nucleus and RNA-associated to be exclusively reactive in PCA serum samples compared to serum from healthy controls10. Beside the diagnostic utility, the autoimmune response towards PCA seems to have also prognostic value. Reactivity towards PSA, Her211 and Fetuin-A12 has been reported to be enhanced in late stages of PCA compared to early. Moreover, autoantibodies in PCA have been reported to be associated with therapy failure in patients treated with androgen deprivation and radiation therapy13. With the aim to identify additional autoantibodies correlating with PCA prognosis, we profiled the IgG repertoire of 589 plasma and serum samples from PCA patients with early and late-stage disease plus 20 controls, starting from 3768 antigens on protein microarrays. The antigens were derived from the Human Protein Atlas (HPA) that generates antibodies specific for each human protein using antigens selected within regions with low homology to other proteins14, 15. These antigens are immobilized on microarray primarily for antibody validation, but can also be used to screen the autoimmune reactivity16, 17. In previous efforts, this approach identified autoantibodies in multiple sclerosis18. In our study, we identified and further characterized the binding region for reactive antigens in serum and plasma in the context of PCA to describe a novel peptide epitope targeted by the immune system. This analysis showed that anti-prostein antibodies in serum and plasma may also be detected in females, which could relate to high homology of the prostein peptide with sequences found in TGIF2 and TMEM79.


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EXPERIMENTAL PROCEDURES Patients and samples Heparin-plasma and serum samples were collected from 609 individuals enrolled in studies at Karolinska University Hospital (Stockholm, Sweden) and Skåne University Hospital (Lund, Sweden). Study sets obtained from these cohorts were respectively denoted set 1 and set 2 (Table 1). For study set 1, serum samples were collected before diagnosis from 20 PCA patients and 20 controls. For study set 2, 569 plasma samples were collected from untreated PCA patients at diagnosis19. Clinical information accompanied samples (e.g. age, gender, cancer stage, Gleason grading, PSA levels, prostate TRUS-volume, ALP and Kreatinin concentration). Samples from PCA patients were classified in High T-stage (T3 or T4) and Low T-stage (T1 or T2), and High Gleason score (>7), Median Gleason score (7) and Low Gleason-score (7) and low (0.1 >0.1 >0.1 >0.1 >0.1 4.94 x 10-6* 2.92 x 10-6* 6.54 x 10-6* 7.81 x 10-5* 0.006* >0.1 >0.1 >0.1 >0.1 0.004* >0.1 >0.1 >0.1 >0.1 >0.1 >0.1 0.011* 0.003*

§ P1 was excluded from the analysis due to a technical reasons. * Intensity levels were statistically different between reactive vs non-reactive.



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Mapping of essential residues of prostein epitopes Next, the epitope scanning analysis was extended on the same samples from set 2 tested for epitope identification. Samples were tested for reactivity towards peptide P10A representing prostein epitope and peptides P10A-A1 – P10A-A9, each representing substitution with alanine or serine of 1 aa within the epitope sequence (Supplementary Table 1). This revealed that not all the residues contribute in the same degree to the epitope recognition. Samples reactive towards the non-modified epitope maintained the same reactivity also towards peptides where alanine was used to substitute the guanine in position 1, the proline in position 2 or the guanine in position 5 respectively. On the contrary, alanine or serine substitutions of amino acids in position 3, 4, 6, 7, 8 or 9 cause a significant (p

Identification of a Novel Autoimmune Peptide Epitope of Prostein in

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