Cervical Cancer Cell Line Secretome Highlights the Roles of Transforming Growth Factor-Beta-Induced Protein ig-h3, Peroxiredoxin-2, and NRF2 on Cervical Carcinogenesis

Abstract

Cancer cells acquire unique secretome compositions that contribute to tumor development and metastasis. The aim of our study was to elucidate the biological processes involved in cervical cancer, by performing a proteomic analysis of the secretome from the following informative cervical cell lines: SiHa (HPV16+), HeLa (HPV18+), C33A (HPV−), and HCK1T (normal). Proteins were analyzed by 2D gel electrophoresis coupled to MALDI-TOF-MS. Enrichment of secreted proteins with characteristic profiles for each cell line was followed by the identification of differentially expressed proteins. Particularly, transforming growth factor-beta-induced protein ig-h3 (Beta ig-h3) and peroxiredoxin-2 (PRDX2) overexpression in the secretome of cancer cell lines was detected and confirmed by Western blot. Bioinformatics analysis identified the transcription factor NRF2 as a regulator of differentially expressed proteins in the cervical cancer secretome. NRF2 levels were measured by both Western blot and Multiple Reaction Monitoring (MRM) in the total cell extract of the four cell lines. NRF2 was upregulated in SiHa and C33A compared to HCK1T. In conclusion, the secreted proteins identified in cervical cancer cell lines indicate that aberrant NRF2-mediated oxidative stress response (OSR) is a prominent feature of cervical carcinogenesis.

Proteomic Analysis of Normal and Cancer Cervical Cell Lines Reveals Deregulation of Cytoskeleton-associated Proteins

“…Most of the proteomic studies utilizing cervical cancer cell lines, focus on the differences that occur in the protein expression pattern as an effect of a drug treatment, such as doxorubicin, oxymatrine and cisplatin (43)(44)(45), or under stress conditions, like UVB irradiation and hypoxia (46,47), or after the induced and/or inhibited expression of specific genes, as in the cases of HVP16 E6 gene, transgelin-2 and parkin (48)(49)(50). Moreover, our group has recently studied the secretome of these cervical cancer cell lines compared to normal cervical keratinocytes (8). Comparative analysis of the secretome revealed 67 differentially expressed proteins, out of which 36 were also identified as differentially expressed in our study.…”

“…Therefore, utilization of informative cell lines with or without the HPV genome, can provide valuable insights on these mechanisms. To this end, our group has initiated a comprehensive approach to elucidate the specific oncogenic drivers operating in cervical cancer, both at the transcriptional (7) and the proteomic level (8). In these studies, we have identified for the first time, four novel transcription modules (7), involved in cervical cancer, exhibiting synergy between groups of transcription regulators, while certain modules were annotated to specific biological processes, such as cell cycle, apoptosis, transcription and development.…”

“…In these studies, we have identified for the first time, four novel transcription modules (7), involved in cervical cancer, exhibiting synergy between groups of transcription regulators, while certain modules were annotated to specific biological processes, such as cell cycle, apoptosis, transcription and development. In our recent study (8), by employing proteomic approaches on the secretome of four informative cervical cell lines, we have identified 67 differentially expressed proteins, displaying mainly catalytic, binding or structural molecule activity, while bioinformatics analysis identified the transcription factor NRF2 as an important regulator of differentially expressed proteins in the cancer cell lines. Thus, such comparative proteomic approaches employing cervical cell lines, represent a valuable tool to further explore the precise mechanisms involved in viral infection and protein dysfunction interplay that can lead to cervical carcinogenesis (5,6).…”

High Resolution Proteomic Analysis of the Cervical Cancer Cell Lines Secretome Documents Deregulation of Multiple Proteases

“…ΗCK1T cells were a kind gift of Dr. Tohru Kiyono (13), and were cultured as proposed (13), in Defined Keratinocyte Serum-Free Medium (Gibco BRL, San Francisco, CA, USA), supplemented with 5 ng/ml Epidermal Growth Factor (Gibco BRL) and 50 μg/ml of Bovine Pituitary Extract (Gibco BRL). The collection of secretome as well as the total cell extract was performed as previously described by our group (12,15).…”

“…Furthermore, the secretome can be a source of putative biomarkers that can be easily detected in biological fluids, such as serum of cancer patients (10,11). We have recently compared the secretome of three representative cervical cancer cell lines (12), such as the C33A (HPV negative), SiHa (HPV16 + ), and HeLa (HPV18 + ) cell lines, to a normal cervical keratinocyte cell line, HCK1T (13), by employing two-dimensional gel electrophoresis coupled to MALDI-TOF mass spectrometry. This initial analysis revealed aberration of several canonical pathways involving metabolic processes and the activation of NRF2-mediated oxidative stress response in cervical cancer cells (12).…”