Cervical cancer is the third most common malignancy in women worldwide. Membrane proteins are involved in cell signaling, cell-cell interactions and transportation. Despite the critical biological significance of membrane proteins, proteomic analysis has been a challenging task, due to their particular biochemical properties. Furthermore, dysfunction of membrane proteins has been shown to correlate with the malignant phenotype in several cancers. Therefore, their systematic study could lead eventually to the discovery of novel drug targets and biomarkers for prognostic or diagnostic purposes. The aim of this study was to compare the expression pattern of membrane proteins of one normal (HCK1T) and three cervical cancer cell lines, i.e. C33A (HPV-), SiHa (HPV16+) and HeLa (HPV18+), in order to discover proteins which may constitute potential biomarkers for cervical cancer.
The procedure used for membrane fraction preparation, involved differential centrifugation and detergent-based solubilization, followed by trypsinization. Peptides were fractionated and identified by high resolution LC-MS/MS. Differentially expressed proteins in cancer cell lines relative to HCK1T, exhibited >2 or <0.5 fold change with a p<0.05 (Mann-Whitney test).
An efficient and reproducible enrichment protocol for membrane proteins was developed. The percentage of membrane proteins in membrane extracts was within the range of 38.0%-42.5% and the percentage of transmembrane proteins within a range of 20.2%- 24.3%. These percentages were significantly higher compared to the total cell extracts. A significant number of unique membrane and transmembrane proteins were identified in the membrane extracts. The average fold enrichment for membrane proteins compared to the total cell extracts was 1.46 and for transmembrane proteins 2.21. The proteomic analysis revealed a variety of differentially expressed membrane proteins involved in signaling pathways associated to cancerogenesis. The identified proteins included Vesicle transport protein GOT1B (testicular seminomas and ovarian carcinoma), GTP-binding protein SAR1b (liver cancer) and ATP-dependent RNA helicase DDX3X (hepatocellular carcinoma).
Unique membrane protein identifications can offer insights on a previously inaccessible part of the cell proteome. Therefore, the successful isolation of membrane proteins in this study generated a significant pool of potential cervical cancer biomarkers and novel promising drug targets. These can be functionally tested in vitro utilizing gene editing approaches, such as the CRISPR/Cas9 system.