Proteomics Strategies to Analyze HPV-Transformed Cells: Relevance to Cervical Cancer

Fabio Di Domenico; Federico De Marco; Marzia Perluigi


Expert Rev Proteomics. 2013;10(5):461-472. 

In This Article

Proteomics Studies in HPV Transformed Cells

The possibility to stably transfect immortalized epithelial cells with specific HPV oncogenes, offers a useful model to investigate the oncogenic transformation driven by papillomavirus infection. Several studies have been carried out by following this approach and contribute to broaden the knowledge on the transforming mechanisms crucial for cervical cancer development.

An initial study from Kyung-Ae Lee et al., was performed to investigate both genes and proteins that may be involved in the immune escape and oncogenesis related mechanism of HPV-transformed cells.[45] In order to achieve this goal, a HPV-negative cervical cancer cell line, C33A, was used to establish a stable cell line expressing E7 and by coupling 2DE and silver staining a comparison of protein expression between mock vector and E7-transfected cells was performed. In parallel, a genomic approach using DNA microarrays was also carried out to unravel which genes could be modulated by the E7 oncogene. Proteins of the cytoskeletal system including actin and myosin V, the tumor suppressor integrase interactor 1 protein (INI) and protein disulfide isomerase A3 were found to be expressed at lower levels in cells expressing E7. INI, a member of the ATP-dependent SWI/SNF chromatin remodeling complex,[46] is involved in the regulation of cell cycle[47] and it is likely that once downregulated may trigger to oncogenic transformation. PDI, catalyzes the formation and isomerization of disulphide bonds; however it was also demonstrated to be involved in cell adhesion and in cell-cell interaction.[48,49] Since previous studies demonstrated that PDI levels markedly increased in apoptotic cells,[50] it is reasonable to hypothesize that E7-induced repression of PDI expression may favor the survival of immortalized cells.

Ku70BP and HSP60 proteins were upregulated by E7. Ku70 plays a role in double-stranded DNA break repair and maintenance of telomeres[51] and E7-induced upregulation of Ku70BP could contribute to its activity.

HSPs are a wide and ubiquitous family of proteins with multiple functions essential for maintaining correct cellular homeostasis.[52,53] In normal cells, HSP60 is constitutively expressed and localizes in the mitochondrial matrix and outer mitochondrial membrane. Usually, it is induced by stress conditions such as heat shock and mitochondrial damage. Cappello et al., also found elevated expression of HSP60 in early and advanced stages of cervical cancer, as well as in prostate cancer.[54] Considering that inhibition of apoptotic machinery is a major clue of cancer progression, upregulation of HSP60 may represent a protective response against cancer development. In addition, Castle et al., showed by immunohistochemistry the upregulation of HSPs in response to HPV infection and proposed the role of HSP60 and HSP70 as surrogate marker for pre-cancer.[55] All together, these results support the possibility that increased expression of HSP60 may play a role as a prognostic factor of cancer development.

In conclusion, it is likely that E7 can escape immune surveillance and avoid apoptosis by the inhibition/induction at the mRNA or protein level of several factors involved in immuneregulation, cell signaling and cell cycle regulation.

These initial findings were further extended by Kyumg-Ae Lee et al.[26] In this study, HaCaT normal keratinocytes were arranged to stably expressing E7 and an E7-affinity column was used to investigate putative target molecules regulated by E7 expression. This experimental approach allowed to identify E7-interacting proteins, among which actin and leukocyte elastase inhibitor were found downregulated and stress-induced phosphoprotein 1, CD2 binding protein 1, catalase, T-complex protein 1, Ku70-binding protein, HSP60, G1/S-specific cyclin E1 and peroxiredoxin 2 were found to be upregulated. Western blot was also carried out to confirm some of the proteomics data for HSP60, catalase and peroxiredoxin 2. An additional pull down assay also demonstrated that leukocyte elastase inhibitor and Ku70-BP interacted with E7.

Overall these data, in agreement with the previous study performed on C33A cells suggested that E7 could influence cell status by modulating the activity of proteins related to intracellular signaling, apoptosis and cell cycle.

A comprehensive overview was achieved by the study of Higareda-Almaraz et al.[56] Six cervical cancer cell lines including two cell lines positive for HPV type 18 (HeLa and CaLo), two positive for HPV type 16 (SiHa and CasKi) and two HPV negative (ViBo and C-33A) and HaCaT, a spontaneously immortalized keratinocyte cell line were analyzed by 2-DE followed by MALDI-TOF. A group of proteins was selected as 'the central core of cervical cancer', that is the common proteomic profile of both positive and negative for HPV differing from the profile corresponding to the non-tumorgenic cell line, HaCaT. Most of the identified proteins are involved in different stages of tumor development and progression as other previous studies have already linked their aberrant expression to the neoplastic phenotype. In detail, the first group of proteins can be grouped and related to cell migration and metastasis, like annexin 2; protein disulphide isomerase; vimentin, an intermediate filament cytoskeletal protein and a marker of epithelial mesenchymal transition and finally vinculin. In a second group, protein members of the Unfolded Proteins Response (UPR), including GRP78, HSP71, HSP7C, HS90B and GRP75 were found. These proteins are activated in response to apoptotic stimuli and therefore promote survival, cell proliferation and angiogenesis.[57] In the last group, alteration of energy metabolism, a well-known phenomenon in cancer biology, was evidenced by the finding of glyceraldehyde 3 phosphate dehydrogenase, phosphoglycerate mutase 1, enolase A, triosephosphate isomerase and L-lactate dehydrogenase B. Accordingly, cancerous cells show a higher glucose intake, increased rate of glycolysis and lactate production which are necessary to support cell growth and DNA replication.

Interestingly, highly proliferative cancer cells are also characterized by increased ROS production and a number of reports suggest that the oncogenic phenotype of cancer cells may result by ROS-induced aberrant intracellular signaling.[58] Indeed, the levels of 8-OHdG (a marker of DNA damage) increase from normal to SIL to invasive carcinomas.[59] In addition to DNA damage, our laboratory[60] showed that keratinocyte cells transfected with the whole HPV-16 genome upon UVB irradiation, showed increased oxidation of a set of proteins which likely lead to their dysfunctions and may play a role in tumor development. However, transformed cells keep the ability to defend themselves from ROS attack through the upregulation of antioxidant system, creating a paradox of high ROS production in the presence of high antioxidant levels.[61] Concurrently, upon UVB irradiation, HPV-transformed cells suppress the viral transcription, activate repair mechanisms ultimately resulting in mild induction of apoptosis.[62] This response was not observed in HaCaT cells that in contrast appeared extremely susceptible to apoptosis. These results led to hypothesize that the suppression of viral oncogenes is part of a protective mechanism that allows the cells to activate repair mechanisms, to control growth pathways ultimately resulting in a surviving response.


Results obtained on cell culture models highlight that HPV oncogenes are able to regulate different cellular functions including cell growth, apoptosis and antioxidant system ultimately resulting in a surviving response that promote the selection of those cell phenotypes with 'transforming' ability.