Spatial Proteomic Analysis of Isogenic Metastatic Colorectal Cancer Cells Reveals Key Dysregulated Proteins Associated with Lymph Node, Liver, and Lung Metastasis

Abstract

Metastasis is the primary cause of colorectal cancer (CRC) death. The liver and lung, besides adjacent lymph nodes, are the most common sites of metastasis. Here, we aimed to study the lymph nodes, liver, and lung CRC metastasis by quantitative spatial proteomics analysis using CRC cell-based models that recapitulate these metastases. The isogenic KM12 cell system composed of the non-metastatic KM12C cells, liver metastatic KM12SM cells, and liver and lung metastatic KM12L4a cells, and the isogenic non-metastatic SW480 and lymph nodes metastatic SW620 cells, were used. Cells were fractionated to study by proteomics five subcellular fractions corresponding to cytoplasm, membrane, nucleus, chromatin-bound proteins, and cytoskeletal proteins, and the secretome. Trypsin digested extracts were labeled with TMT 11-plex and fractionated prior to proteomics analysis on a Q Exactive. We provide data on protein abundance and localization of 4710 proteins in their different subcellular fractions, depicting dysregulation of proteins in abundance and/or localization in the most common sites of CRC metastasis. After bioinformatics, alterations in abundance and localization for selected proteins from diverse subcellular localizations were validated via WB, IF, IHC, and ELISA using CRC cells, patient tissues, and plasma samples. Results supported the relevance of the proteomics results in an actual CRC scenario. It was particularly relevant that the measurement of GLG1 in plasma showed diagnostic ability of advanced stages of the disease, and that the mislocalization of MUC5AC and BAIAP2 in the nucleus and membrane, respectively, was significantly associated with poor prognosis of CRC patients. Our results demonstrate that the analysis of cell extracts dilutes protein alterations in abundance in specific localizations that might only be observed studying specific subcellular fractions, as here observed for BAIAP2, GLG1, PHYHIPL, TNFRSF10A, or CDKN2AIP, which are interesting proteins that should be further analyzed in CRC metastasis.

Identification of TRAPPC9 and BAIAP2 Gene Polymorphisms and Their Association With Fat Deposition-Related Traits in Hu Sheep

“…The expression of BAIAP2 in the liver was significantly higher than that in other tissues (P < 0.05). Previous studies have found that the BAIAP2 is significantly expressed in the liver (37)(38)(39). The liver plays a pivotal role in regulating the metabolism of fatty acids (FA) and their neutral storage form, triglycerides (TGs) (40,41).…”

Proteomic and in silico analyses of dextran synthesis influence on Leuconostoc lactis AV1n adaptation to temperature change

“…Then, a two-sample Student’s t -test with a permutation-based FDR was performed. Only proteins with an adjusted p -value ≤ 0.05, FDR 5%, and log2 ratio of ≥ 1.5 or ≤ 0.67 were considered as deregulated following the same criteria as in other proteomic analyses ( Peláez-García et al, 2015 ; Liu et al, 2017 ; Romero-Gavilán et al, 2022 ; Solis-Fernández et al, 2022 ). The protein identification by nLC-MS/MS was carried out in the Proteomics and Genomics Facility at the Biological Research Center Margarita Salas (CIB-CSIC).…”

Metabolic Reprogramming Helps to Define Different Metastatic Tropisms in Colorectal Cancer

“…Then, the protein extracts were centrifuged for 10 min at 4°C at 10,000 g, and supernatants were transferred to new tubes. The protein concentration was determined by tryptophan quantification ( 22 , 26 , 27 ) and confirmed by Coomassie blue staining. Five micrograms of each protein extract was separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and transferred to nitrocellulose membranes.…”