Alteration of protein expression and spliceosome pathway activity during Barrett’s carcinogenesis

J Gastroenterol (2021) 56:791–807 https://doi.org/10.1007/s00535-021-01802-2 ORIGINAL ARTICLE—ALIMENTARY TRACT Alteration of protein expression and spliceosome pathway activity during Barrett’s carcinogenesis Christoph Stingl1 • Angela Bureo Gonzalez2 • Coşkun Güzel1 • Kai Yi Nadine Phoa2 • Michail Doukas3 • Gerben Eise Breimer4,5 • Sybren Lodewijk Meijer4 • Jacques Johannes Bergman2 • Theo Marten Luider1 Received: 6 November 2020 / Accepted: 18 June 2021 / Published online: 5 July 2021 Ó The Author(s) 2021 Abstract Background Barrett’s esophagus (BE) is a known precursor lesion and the strongest risk factor for esophageal adenocarcinoma (EAC), a common and lethal type of cancer. Prediction of risk, the basis for efficient intervention, is commonly solely based on histologic examination. This approach is challenged by problems such as interobserver variability in the face of the high heterogeneity of dysplastic tissue. Molecular markers might offer an additional way to understand the carcinogenesis and improve the diagnosis—and eventually treatment. In this study, we probed significant proteomic changes during dysplastic progression from BE into EAC. Methods During endoscopic mucosa resection, epithelial and stromal tissue samples were collected by laser capture microdissection from 10 patients with normal BE and 13 patients with high-grade dysplastic/EAC. Samples were analyzed by mass spectrometry-based proteomic analysis. Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/s00535021-01802-2. & Christoph Stingl 1 Department of Neurology, Erasmus University Medical Center, PO Box 20440, 3000 CA Rotterdam, The Netherlands 2 Department of Gastroenterology and Hepatology, Amsterdam University Medical Centers, Amsterdam, The Netherlands 3 Department of Pathology, Erasmus University Medical Center, Rotterdam, The Netherlands 4 Department of Pathology, Amsterdam University Medical Centers, Amsterdam, The Netherlands 5 Present Address: Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands Expressed proteins were determined by label-free quantitation, and gene set enrichment was used to find differentially expressed pathways. The results were validated by immunohistochemistry for two selected key proteins (MSH6 and XPO5). Results Comparing dysplastic/EAC to non-dysplastic BE, we found in equal volumes of epithelial tissue an overall up-regulation in terms of protein abundance and diversity, and determined a set of 226 differentially expressed proteins. Significantly higher expressions of MSH6 and XPO5 were validated orthogonally and confirmed by immunohistochemistry. Conclusions Our results demonstrate that disease-related proteomic alterations can be determined by analyzing minute amounts of cell-type-specific collected tissue. Further analysis indicated that alterations of certain pathways associated with carcinogenesis, such as micro-RNA trafficking, DNA damage repair, and spliceosome activity, exist in dysplastic/EAC. Keywords Barrett’s esophagus  Adenocarcinoma  Laser capture microdissection  Mass spectrometry  Proteomics Abbreviations AGC Automatic gain control APA Alternative polyadenylation BE Barrett’s esophagus DSB Double-strand breakage EAC Esophageal adenocarcinoma ELISA Enzyme-linked immunosorbent assay EMR Endoscopic mucosa resection ER-cap Endoscopic resection cap technique ESI Electrospray ionization FDR False discovery rate FFPE Formalin fixed and paraffin embedded 123 792 GERD GO HE HGD iBAQ IHC LC–MS LC LCM LFQ LGD MALDI MMR MS/MS NDBE NHEJ SNP TMA u UTR J Gastroenterol (2021) 56:791–807 Gastroesophageal reflux disease Gene ontology Hematoxylin and eosin High-grade dysplasia Intensity-based absolute quantification Immunohistochemistry Liquid chromatography coupled to mass spectrometry Liquid chromatography Laser capture microdissection Label-free quantification Low-grade dysplasia Matrix-assisted laser desorption/ionization Mismatch repair genes Tandem (or fragment) mass spectrum Non-dysplastic Barrett’s epithelium Non-homologous end joining Single-nucleotide polymorphism Tissue micro-array Atomic mass unit Untranslated region Introduction In Barrett’s esophagus (BE), the normal squamous lining of the lower esophagus is replaced by gastric type columnar epithelium [1]. This condition is considered a consequence of chronic gastroesophageal reflux disease (GERD). Because BE is asymptomatic, it is most commonly diagnosed by endoscopy in patients with GERD symptoms [2]. It is, therefore, difficult to assess the prevalence for the general population, and a biased group of patients undergo endoscopy because of symptoms that are not necessarily related to BE [3]. Dependent on the scope and population of a study, the reported average prevalence of histologically confirmed BE is around 1.5% (0.1–9.0%) [3–5]. BE is considered a premalignant precursor for esophageal adenocarcinoma (EAC), which might progress continuously through the sequence of low-grade dysplasia (LGD), highgrade dysplasia (HGD) and ultimately adenocarcinoma. It follows that both non-dysplastic BE and dysplastic BE are important risk factors for EAC [6]. The prognosis of EAC is poor; the 5-year survival rate is low, at 10–18% dependent on sex and ethnicity [7, 8]. EAC occurs predominately in males, with the highest rates in Western and Central Asia regions [9, 10], and is currently the sixth most frequent cancer, with the highest increase of incidence rate in the past 3 decades [11]. BE is diagnosed by the presence of endoscopically visible and histopathologically confirmed metaplasia [12]. 123 The grade of dysplasia is strongly related to the risk of carcinogenesis [13] and defines the intensity of the required surveillance and treatment [14]. However, distinguishing between different grades of dysplasia is challenging and in the past resulted in low inter-observer agreement and variation in the assessment of risk of progression between studies [15]. As a consequence, the risk prediction of EAC solely on basis of the dysplastic grade is of limited reliability, potentially may lead to overtreatment [16]. The pathological progression from BE into EAC is associated with biological processes such as proliferation, tumor suppression, cell adhesion and inflammation. Molecules involved in these pathways might predict the development of EAC. A wide range of molecular markers have been studied, such as genomic alterations, epigenetic markers and proteins expression[17–19]: DNA copy number variations and aneuploidy have been found to be altered in EAC [20], and regions of loss of heterozygosity have been identified as promising predictive markers for EAC [21]. Gains of chromosomes 7 and 17 determined by FISH have been found correlated with the grade of oncogenic progression; the detection rate of dysplasia improved when the assessment of these gains was added to cytology [22]. EAC is char (...truncated)