Project description:Gene expression profiling has been widely used to screen for metastasis-associated genes by comparing the difference in paired primary and metastatic colorectal carcinomas, orthotopic implantation mouse model or cells with different metastatic potential in the field of CRC research In our study, we observed that the genes differentially expressed in M5 relative to its parent SW480 cell line Here we used the SW480, a human CRC cell line, as a model system. In vivo variant lines with increasing metastatic capacity were selected in a metastatic orthotopic model of human CRC. A subpopulation named as M5 with enhanced metastatic abilities to liver was isolated by in vivo selection of SW480 cells. We identified genes associated with tumor metastasis by comparing the difference between high metastatic subclone and its parent cells.
Project description:Understanding cancer metastasis at the proteoform level is crucial for discovering new protein biomarkers for cancer diagnosis and drug development. Proteins are the primary effectors of function in biology and proteoforms from the same gene can have drastically different biological functions. Here, we present the first qualitative and quantitative top-down proteomics (TDP) study of a pair of isogenic human metastatic and non-metastatic colorectal cancer (CRC) cell lines (SW480 and SW620). This study pursues a global view of human CRC proteome before and after metastasis in a proteoform-specific manner. We identified 23,319 proteoforms of 2,297 genes from the CRC cell lines using capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS), representing nearly one order of magnitude improvement in the number of proteoform identifications from human cell lines compared to literature data. We identified 111 proteoforms containing single amino acid variants (SAAVs) using a proteogenomic approach and revealed drastic differences between the metastatic and non-metastatic cell lines regarding SAAVs profiles. Quantitative TDP analysis unveiled statistically significant differences in proteoform abundance between the SW480 and SW620 cell lines on a proteome scale for the first time. Ingenuity Pathway Analysis (IPA) disclosed that many differentially expressed genes at the proteoform level had diversified functions and were closely related to cancer. Our study represents a milestone in TDP towards the definition of human proteome in a proteoform-specific manner, which will transform basic and translational biomedical research.
Project description:As one of the leading causes of cancer deaths worldwide, colorectal cancer (CRC) development is closely associated with the accumulation of both genetic and epigenetic alterations. Many efforts have been made to investigate the role of epigenetic modifications in CRC metastasis. In this work, we present the first quantitative top-down proteomics study focusing on histone proteoforms between metastatic (SW620) and nonmetastatic (SW480) CRC cells to reveal potentially critical histone proteoforms in CRC metastasis. We isolated histone proteins from CRC cells, separated them by sodium dodecyl-sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), recovered each histone protein from the gel, and analyzed them by capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS). A total of 230 histone proteoforms were quantified in SW480 and SW620 cell lines, among which 34 proteoforms were significantly altered in abundance in the metastatic cells, indicating a significant transformation of histone proteoforms during metastasis. We observed a significant increase in abundance of all nine differentially expressed histone H4 proteoforms in metastatic SW620 cells compared to SW480 cells, while differentially expressed proteoforms of other histone proteins display diversified expression patterns. Additionally, two histone H2A proteoforms with a combination of N-terminal acetylation and phosphorylation were upregulated in the metastatic CRC cells. These differentially expressed histone proteoforms could be novel proteoform biomarkers of CRC metastasis.
Project description:Gene expression profiling has been widely used to screen for metastasis-associated genes by comparing the difference in paired primary and metastatic colorectal carcinomas, orthotopic implantation mouse model or cells with different metastatic potential in the field of CRC research In our study, we observed that the genes differentially expressed in M5 relative to its parent SW480 cell line