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.
Highlights
Colorectal cancer (CRC), with an estimated overall incidence in the general population of 447 per 100,000, is the second most common cancer in Europe after lung cancer [1,2]
We focused our attention on interesting proteins not previously associated with CRC or CRC metastasis and whose information in databases was scarce, such as brain-specific angiogenesis inhibitor 1-associated protein 2 (BAIAP2), Golgi apparatus protein 1 (GLG1), phytanoyl-CoA 2-hydroxylase-interacting protein-like (PHYHIPL), tumor necrosis factor receptor superfamily member 10A (TNFRSF10A), and CDKN2A-interacting protein (CDKN2AIP), in contrast to other proteins, such as SCRIB, SNX9, CLDN3, or AHR, which have been previously associated with CRC and/or CRC metastasis [47,48,49,50]
We provided a comprehensive proteomic analysis of CRC metastasis using isogenic CRC cell lines with different metastatic tropisms, identifying several proteins and pathways upregulated in CRC metastasis
Summary
Colorectal cancer (CRC), with an estimated overall incidence in the general population of 447 per 100,000, is the second most common cancer in Europe after lung cancer [1,2]. The use of different schedules of chemotherapies combined with targeted biological therapies has considerably improved the median overall survival for patients with metastatic CRC [1]. The majority of patients with metastatic CRC progress in spite of their initial treatment receiving second and third line treatments, which results in a less than 10% 5-year survival probability. Cancer cells that are able to survive in the circulation will be able to reach a secondary tissue by extravasation and colonization [6]. All of these events where cancer cells need to interact with different microenvironments possess unique molecular characteristics [7,8]. There, cancer cells must adapt to each situation, altering the expression, localization, and activation of proteins to generate a metastasis
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