Cervical cancer results from infection by certain HPV types. Specifically, HPV types 16 and 18 are responsible for 70% of cervical cancers and precancerous cervical lesions. The aim of the study was to identify proteins and biological processes relevant to cervical malignancy by proteomic analysis of the secretome from normal and cancer cell lines.
Secreted proteins were collected from three representative cervical cancer cell lines, SiHa (HPV16+), HeLa (HPV18+), C33A (HPV-) and HCK1T a normal cervical epithelium cell line which served as control. The secretome samples were analyzed by high resolution mass spectrometry LC/MS-MS and evaluated by Proteome Discoverer 1.4, while only high peptide confidence identifications were accepted. The proteins with statistically significant (Mann Whitney p<0.05) differential expression (fold change from >2 to <0.5), were subjected to bioinformatics analysis by the Ingenuity Pathway Analysis (IPA) software.
The LC/MS-MS analysis identified 1200-1300 secreted proteins for each cell line, a significantly higher number compared to the 300-400 proteins identified by 2D electrophoresis coupled to MS. A number of proteins with important biological functions were identified, such as the extracellular matrix remodeling proteases Cathepsins, which were upregulated in cancer cell lines (SiHa, HeLa, C33A) vs normal (HCK1T). Bioinformatic analysis revealed significant pathways deregulated in cervical cancer such as Inhibition of Matrix Metalloproteases (MMPs) and oxidative stress response. The proteomics results and bioinformatic predictions were validated in cervical cell lines and in clinical samples. High levels of Cathepsin D, a secreted protease that plays a pivotal role in remodeling and degradation of extracellular matrix, were detected in cancer samples by Western blot. Moreover, a cervical cancer-associated increase in MMPs enzymatic activity was determined by zymography assays. The under-expression of anti-oxidant enzyme Superoxide Dismutase 2 (SOD2) in cervical malignancy was confirmed by Western blot.
The systems biology approach used to study cervical cancer resulted in the discovery and subsequent validation of significant proteins and pathways that play important roles in malignancy. The most prominent findings included: proteases secreted by cancer cells, such as Cathepsin D and MMPs, which are involved in the process of invasion of the surrounding tissues, and enzymes implicated in oxidative stress response, such as SOD2.