Study Of Serum Peptides Markers And Transcriptional Regulation Of ABCC4 In Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant neoplasms worldwide. Patients are often diagnosed at advanced stages with poor prognoses due to the absence of obvious early symptoms. The average 5 -year survival rate of ESCC are within 30-40%, despite early stage ESCC exceeding 90%. Importantly, there are no established serological tumor markers for ESCC currently.Firstly, we measured the concentration of serum Cyfra21-1 and squamous cell carcinoma antigen (SCC) in patients with ESCC and dysplasia lesions, and healthy controls by enzyme-linked immunosorbent assay (ELISA). The levels of serum Cyfra21-1 in ESCC and dysplasia patients were significantly higher than those in healthy controls (P<0.05). Significant statistical difference was found in multiple comparison (P<0.05) for SCC. The expression of Cyfra21-1 was higher in patients with positive lymphatic metastasis (P=0.005). However, the correlation between Cyfra21-1 and SCC expression and the other histopathological features such as age, gender, position, pathological grade, tumor size and TNM staging was not observed in our samples. According to receiver operating characteristic curve (ROC) curve, the combine of Cyfra21-1 and SCC was much better than each of them. The results of electrochemiluminescence immunoassay (ECLIA) and micro particle Enzyme Immunoassay (MEIA) of Cyfra21-1 and SCC were similar with the ELISA results.Next, we applied a high-throughput serum peptidome analysis to identify circulating peptide markers of ESCC. Weak cationic exchange magnetic beads coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used for two-stage proteotypic peptide profiling in complex serum samples collected from 477 cancer patients and healthy controls. We established a genetic algorithm model containing three significantly differentially expressed peptides at 1,925.5,2,950.6 and 5,900.0 Da with a sensitivity and specificity of 97.00%(97/100) and 95.92%(94/98)in the training set and 97.03%(98/101) and 100.00%(98/98) in the validation set, respectively. The model's diagnostic capability was significantly better than SCC and Cyfra 21-1, especially for early stage ESCC, with an achieved sensitivity of 42/43(97.67%). Subsequently, these peptides were identified as fragments of Alpha2-HS glycoprotein (AHSG), Thrombospondin-1 (TSP1) and Fibrinogen A alpha-chain (FGA) by linear ion trap-orbitrap hybrid tandem mass spectrometry. Notably, increased tissue and serum levels of TSP1 in ESCC were verified and correlated with disease progression. In addition, tissue TSP1 was an independent poor prognostic factor in ESCC.Thirdly, because the N-terminal peptide of TSP1 was overexpressed in the sera of ESCC patients, we designed and produced an antibody that specifically recognized the fragment of TSP1. Furthermore, a competitive ELISA assay for specific detection of the peptide was established. The preliminary experiments showed that the median sera levels of TSP1 in ESCC patients were significantly higher than those in health controls (P<0.05). The newly established TSP1 antibody and ELISA assay could serve as potential tools for detection and diagnosis of ESCC. TSP1 and its N-terminal fragment may act as a potential biomarker for ESCC. Still, a larger cohort will be required for further unequivocal validation of their perspectives in clinic.Lastly, the previous research in our lab found that the copy number amplification of ATP-binding cassette transporter family class C4 (ABCC4) gene results in overexpression of ABCC4 protein in ESCC, which might involved in tumorigenesis. ABCC4 is known as a member of the ATP-binding cassette transporter family. ABCC4 plays an important role in physiologic, endogenous and exogenous substances transportation, and it was also known as multidrug resistance-associated protein 4 (MRP4). ABCC4 can also reduce the intracellular concentration and the sensitivity of various chemotherapy drugs. Here, we further checked the expression of ABCC4 in ESCC cell lines using Western blot analysis, and then determined the effect of ABCC4 protein levels on therapy response to cisplatin. Compared with ABCC4 low expression cell lines, ABCC4 highly expressed cell lines were more resistant to cisplatin. After knock down ABCC4 in KYSE140, the chemosensitivity was elevated. Furthermore, we narrowed down the core sequence of ABCC4 enhancer using luciferase reporter gene assay and confirmed several transcription factors binding to this region using Chromatin Immunoprecipitation-Polymerase Chain Reaction (ChIP-PCR). These data showed that ABCC4 plays important roles in ESCC chemotherapy and serves as a candidate of chemotherapy resistance. The potential activators and repressors that regulate ABCC4 expression should be further analyzed in ESCC.