Proteomic Characterization of Transcription and Splicing Factors Associated with a Metastatic Phenotype in Colorectal Cancer

Abstract

We investigated new transcription and splicing factors associated with the metastatic phenotype in colorectal cancer. A concatenated tandem array of consensus transcription factor (TF)-response elements was used to pull down nuclear extracts in two different pairs of colorectal cancer cells, KM12SM/KM12C and SW620/480, genetically related but differing in metastatic ability. Proteins were analyzed by label-free LC-MS and quantified with MaxLFQ. We found 240 proteins showing a significant dysregulation in highly metastatic KM12SM cells relative to nonmetastatic KM12C cells and 257 proteins in metastatic SW620 versus SW480. In both cell lines there were similar alterations in genuine TFs and components of the splicing machinery like UPF1, TCF7L2/TCF-4, YBX1, or SRSF3. However, a significant number of alterations were cell-line specific. Functional silencing of MAFG, TFE3, TCF7L2/TCF-4, and SRSF3 in KM12 cells caused alterations in adhesion, survival, proliferation, migration, and liver homing, supporting their role in metastasis. Finally, we investigated the prognostic value of the altered TFs and splicing factors in cancer patients. SRSF3 and SFPQ showed significant prognostic value. We observed that SRSF3 displayed a gradual loss of expression associated with cancer progression. Loss of SRSF3 expression was significantly associated with poor survival and shorter disease-free survival, particularly in early stages, in colorectal cancer.

SWATH‐based proteomics reveals processes associated with immune evasion and metastasis in poor prognosis colorectal tumours

“…40,41 Furthermore, a strong inverse correlation was found between transcriptome instability and the expression of splicing factor genes, which was also associated with poor patient survival. 21,40,41 Our results also indicated in P3 tumours an increased expression of proteins related to acute inflammatory response, wound healing response and complement pathway activation. Zhang et al 42 identified five proteomic subtypes in genomically annotated TCGA colon tumours and reported that the proteomic subtype with stemlike characteristics was enriched in genes involved in wounding response.…”

“…Notably, studies on differential splicing events among tumours support transcriptome instability as a molecular characteristic of CRC . Furthermore, a strong inverse correlation was found between transcriptome instability and the expression of splicing factor genes, which was also associated with poor patient survival …”

“…Different proteomic technologies have been used to find new cancer biomarkers for CRC employed for risk prediction, diagnosis, prognosis, staging and monitoring treatment response of this disease. First proteomic studies focused on two‐dimensional electrophoresis (2‐DE) to characterize diseased vs control proteomes in combination with MALDI‐TOF MS or LC‐MS/MS . Nevertheless, these proteomic approaches involve hydrophobicity, size and solubility limitations of protein samples, and label‐free LC‐MS technologies have been used as an alternative approach in the search of CRC biomarkers .…”

“…First proteomic studies focused on two‐dimensional electrophoresis (2‐DE) to characterize diseased vs control proteomes in combination with MALDI‐TOF MS or LC‐MS/MS . Nevertheless, these proteomic approaches involve hydrophobicity, size and solubility limitations of protein samples, and label‐free LC‐MS technologies have been used as an alternative approach in the search of CRC biomarkers . Nowadays, SWATH technique provides comprehensive and permanent records of all detectable molecular species and this proteomic technology is emerging as a powerful tool for CRC research.…”

“…[19][20][21] Nevertheless, these proteomic approaches involve hydrophobicity, size and solubility limitations of protein samples, 22 and label-free LC-MS technologies have been used as an alternative approach in the search of CRC biomarkers. 21 Nowadays, SWATH technique provides comprehensive and permanent records of all detectable molecular species and this proteomic technology is emerging as a powerful tool for CRC research. Thus, SWATH-MS-based quantitative proteomic analysis has been used to analyse glycoproteins secreted by a colon adenocarcinoma cell line that were confirmed by immunodetection 23 or to determinate the molecular effects induced by DDHD1 silencing in colorectal cancer cells.…”