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 on Human Tissues: CINs & Cervical Carcinoma

Interesting data were also collected by the analyses of human tissues, both CINs and carcinoma biopsies, which allowed to understand both pathogenesis and progression of cervical cancer. Indeed, screening in cervical cancer tissues is progressing to find out candidate genes and proteins, which may work as biological markers for more accurate diagnosis and therapeutic strategies.[1]

The study from Bae et al., analyzed the protein expression patterns of squamous cell carcinoma (SCC) tissues from Korean women using 2DE and MALDI-TOF.[63] A total of 35 proteins were identified to be differently expressed, among which 12 (pigment epithelium derived factor, annexin A2 and A5, keratin 19 and 20, heat shock protein 27, smooth muscle protein 22 α, α-enolase, squamous cell carcinoma antigen 1 and 2, glutathione S-transferase, apolipoprotein a1) were already known to be involved in tumor development. Among these results it worth to be mentioned annexin A2, a Ca2+ and phospholipid-binding protein, found to be up-regulated in agreement with other findings showing its increased expression in other tumors.[64,65] Further, HSP27 was found to be increasingly expressed in SCC compared with control tissues, as already observed in human ovarian and mammary tumors.[66–68] HSP27 is mainly expressed in well-differentiated and moderately differentiated SCCs.[69] Accordingly, HSP27 was found to be upregulated in C33A cell transfected with HPV E7 gene (see above).

Another study from Zhu et al., analyzed proteins from human SCC matched with adjacent normal cervical tissues by 2DE and MALDI-TOF.[25] A comparison of protein profiles revealed a number of proteins that may play a role in tumorigenesis, whereas other proteins could be of interest as potential biomarkers for diagnosis and/or therapy. Among these tyrosine kinase 2 (Tyk2), S100A9 and zinc-finger protein 217 (ZNF217) are the most relevant in tumor biology.

In the current study, Tyk2 was found over-expressed in SCC; with a high incidence in 18 of 20 SCC tissues but only in 2 of 20 matched control tissues. Tyk2, a member of the Jak family of kinases, is known to play important roles in receptor signal transduction by interferon, interleukins, growth factors and other hormones. The study by Ide et al., demonstrated in prostate cancer cells that Tyk2 suppression either by small interfering RNA targeting or by the use of a Tyk2 inhibitor, resulted in enhanced invasiveness of prostate cancer cells in vitro without affecting the cellular growth rate.[70]

S1009 protein was also found to be expressed at higher levels in SCC. S100 calcium-binding proteins are involved in a variety of intracellular pathways mediated by alteration of calcium levels, which regulate cell growth, differentiation, migration, adhesion among others. Increased S100A9 levels were measured in various human cancers.[71–74] Chao et al., showed by DNA microarrays the upregulation of S100A9 gene in squamous carcinoma by comparing carcinoma specimens and the surrounding normal tissue.[75] Further studies are needed to relate the expression of S100A9 with cellular differentiation, as well as invasion and migration.

ZNF217 can affect the amplification of 20q13 during critical early stages of cancer progression thus leading to immortalization of human ovarian and mammary epithelial cells. Using RNA interference, Sun et al., demonstrated that ZNF217 knockdown in ovarian cancer cell line was able to inhibit cancer cell growth and also their invasive potential.[76]

As we previously discussed, HPV infection, although considered the major tumorigenic factor, is not per se sufficient to induce cervical cancer. Recently, growing attention has been directed to clarify the role of oxidative stress in HPV-driven carcinogenesis, but further studies are needed. With this purpose, our group analyzed a small group of samples including HPV-16 positive tissues from patients with invasive squamous cervical carcinoma, from patients with high grade dysplastic HPV lesions and from control patients. First, we evaluated the modulation of the redox status demonstrating the upregulation of ERp57 and GST in dysplastic and neoplastic tissues compared with controls. Further, by applying a redox proteomic we showed increased oxidation (carbonylation) of cytokeratin 6, actin, cornulin, retinal dehydrogenase and GAPDH in dysplastic samples compared to controls. Conversely, carcinoma samples were found less oxidized than dysplastic tissues as indexed by decreased carbonylation of the following proteins: peptidyl-prolyl cis-trans isomerase A, ERp57, serpin B3, annexin 2 and GAPDH. We suggest that oxidative damage accumulate in dysplastic tissues where selective dysfunction of proteins, such those involved in cell morphogenesis and terminal differentiation, may eventually participate, in the presence of additional unknown events, to neoplastic transformation.[21,31]


Proteomics-based approach on human tissue allows the achievement of a comprehensive protein profile of squamous cervical cancer. Compared with in vitro cell models which led to the identification of some common protein targets, results obtained on different cohort of patients instead of being individually considered should be combined (Figure 3). Ongoing analysis of these differential proteins will provide further evidences for their possible implications in squamous cervical cancer-specific diagnosis and therapeutic applications. Further studies on large population, by also including patients at different CIN grades, are needed to extend the limited results obtained so far and also to confirm available data.

Figure 3.

Proteomics approaches to study HPV-transformed cells and cervical cancer. The identified proteins both in vitro and in vivo are mainly involved in cell growth and differentiation (vimentin, keratin19, etc.), cell migration and metastasis (ANX2, PDI, ZNF217) and apoptosis evasion (S100A9, Ku70BP, GRP78). The unique proteins identified in common are ANXs and HSPs.

A discovery phase study by Gu et al., employed a recently developed strategy to identify candidate protein biomarkers for high-grade dysplastic cervical cells from ThinPrep.[77] Laser capture microdissection (LCM) allows isolating high-grade dysplastic and normal cells from ThinPrep slides prepared from cervical cytological specimens. After isolation, proteins can be easily analyzed by gel electrophoresis, digested and analyzed by liquid chromatography mass spectrometry (LC-MS). In total, nine normal (negative for intraepithelial lesions) and nine abnormal cervical cytological specimens high-grade squamous intraepithelial lesion (HSIL) were included and a huge number of proteins showed a threefold difference in protein level, with a significant upregulation of nuclear and mitochondrial proteins in HSIL specimens.

Cytological and molecular screening programs of HPV infection commonly utilize ThinPrep slides. After DNA extraction, hybridization and PCR assay allow to measure HPV DNA copy number and, in some cases, if it correlates with different stages of the cervical disease. ThinPrep has the advantage to contain secreted, shedded, vesicle derived and HPV proteins, and could be an ideal source to search for potential biomarkers of cancer risk. Recently, a study from Papachristou et al., allowed to detect six HPV-derived proteins, including the high-risk HPV16 type, in ThinPrep smear specimens from HPV positive and HPV negative females by a high precision MudPIT, iTRAQ labeling and high-resolution FT-Orbitrap MS. These data were also confirmed with PCR-hybridization molecular methods.[35]

A different approach has been employed to analyze the water-soluble protein fraction from formaldehyde-fixed paraffin embedded punch biopsies by SELDI-TOF MS.[78] The results obtained by this study evidenced that the water-soluble proteins are good candidate to support the diagnosis of normal and CIN2-3 patients. Indeed, CINs are the first type of lesions that can be graded (CIN 1–3) before the development of cervical. SELDI-TOF MS technology has been challenged in many studies to identify new, early and reproducible marker for cancer detection[79,80] or for different lymphomas.[81] One of the most important goal is to verify the ability of this approach, and eventually other novel sophisticated MS platforms, to discriminate between CINs that will regress spontaneously from CINs that will persist or progress to invasive cancer.

An example of new emerging technologies that promise to facilitate high-throughput proteomics is represented by protein macroarrays. This system involves the development of a peptide aptamer microarray that detects endogenous E6 and E7 oncoproteins from different biological samples. HPV16-infected cervical keratinocytes were used to test the ability of differentiating E6 and E7 protein levels in cells containing episomal and integrated HPV16 genomes.[44] In addition to detect low levels of E6 and E7 proteins, the peptide aptamers is able to recognize specific cellular proteins interacting with E6 and E7. By analyzing these interactions, it was suggested that expression of cyclin-dependent kinase 2 (CDK2), CDK4 and BCL-6 is altered in the presence of HPV infection and genome integration. Protein arrays represents a promising approach to quantify the expression levels of HPV oncoproteins in clinical samples and thus to differentiate different clinical stages of lesions.