Abstract
Abstract Background: 5FU is widely used and inhibits thymidylate synthase (TS); LV given in combination with 5FU enhances TS inhibition. TS levels and inhibition are associated with survival in advanced disease. The large variation in response between patients indicates that more mechanisms may be important. To make better treatment decisions, several additional biomarkers, next to TS, such as 5FU activation, breakdown and incorporation into RNA have been investigated. Several studies on global molecular profiling for 5FU activity have been performed on model systems and patients, showing that loss of 18p11-32, which harbors the TS gene, was associated with better survival. Aim: Identification of new potential mechanisms of action and potential biomarkers of 5FU with or without LV treatment using in-depth proteomics analysis of patient CRC tissues. Methods: Patients received approximately 48 hours before resection, a test dose of 5FU (500 mg/m2) (n = 5), 5FU with LV (500 mg/m2) (n = 7) or no test dose (n = 7). For proteomics analysis, samples of CRC resection material were lysed in SDS sample buffer, fractionated by 1D gel electrophoresis, followed by in-gel digestion and nano-liquid chromatography coupled to tandem mass spectrometry (QExactive). The MaxQuant tool was used for protein identification and quantification by spectral counting. Results: Our CRC proteome analysis identified a total of 6880 proteins and 874 with altered abundance (p< 0.05) upon treatments. Our results indicate that 5FU induces upregulation of proteins associated with the extracellular matrix, membrane vesicles, stress and immune responses and downregulates mitochondrial proteins, peroxisomes and (most profoundly) ribosomal proteins. Patients who received 5FU-LV also displayed upregulation of extracellular matrix and vesicle/granule proteins in addition to cell adhesion proteins, an extensive downregulation of ribosomal proteins and downregulation of mitochondrial and ubiquitin proteins. In addition, there is an upregulation of the GO-term ‘Extracellular region part’ in both 5FU and 5FU/LV treated patients, compared to controls. Top upregulated proteins were isoform NELF-D of negative elongation factor C (NELFCD), AMP deaminase 3 (AMPD) and myeloblastin (PRTN3), while top downregulated were neudesin (NENF), antigen KI-67 (MKI67) and HERC4. Conclusions: Proteome analysis revealed strong changes in the CRC proteome upon 48 hrs of 5FU +/- LV treatment, with regulated proteins involved in stress and immune responses, vesicular transport, protein synthesis and metabolism. A number of proteins were not yet associated to 5FU actions. These proteins when correlated in a larger cohort to clinical outcome may function as biomarkers for future treatment decision. Citation Format: Kees Smid, Erik Meijer, Thang V. Pham, Inge de Reus, Sander R. Piersma, Godefridus J. Peters, Connie R. Jimenez. Proteomics analysis of the effect of fluorouracil (5FU) and 5FU/leucovorin (LV) on colorectal cancer (CRC) in patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 572. doi:10.1158/1538-7445.AM2015-572
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